Plant resistant to helminthosporium turcicum

ABSTRACT

The present invention provides an improved Helminthosporium turcicum-resistant plant, in particular a maize plant which comprises a polynucleotide with one or more resistance-conferring genes, for example on a truncated chromosome fragment from the accession Pepitilla, as well as a cell, a tissue, a part, grain and seeds thereof, an isolated polynucleotide which comprises one or more resistance-conferring genes against Helminthosporium turcicum, a vector, a transgenic plant cell and a transgenic plant containing this polynucleotide. Furthermore, the invention encompasses suitable markers and their use in introducing resistance or the transgene into a plant, as well as the identification of improved maize plants which comprise a truncated chromosome fragment.

FIELD OF THE INVENTION

The present invention relates to the field of the modification of plantsusing molecular biological methods and marker technology, along withgenetic engineering. It concerns a novel Helminthosporiumturcicum-resistant plant, in particular a maize plant which comprises apolynucleotide with one or more resistance-conferring genes on amodified chromosome fragment from the accession Pepitilla, as well as acell, a tissue, a portion, grain and seed thereof, an isolatedpolynucleotide which comprises one or more resistance-conferring genesagainst Helminthosporium turcicum, a vector, a transgenic plant cell anda transgenic plant containing this polynucleotide. The invention alsoencompasses suitable molecular markers and their use in introducing theresistance locus or the transgene into a plant, as well as theidentification of improved maize plants which comprise a modifiedchromosome fragment.

BACKGROUND OF THE INVENTION

In maize (Zea mays L.), there are a large number of fungal pathogenswhich cause leaf diseases. The fungus which can cause by far the mostdamage under tropical and also under temperate climatic conditions, suchas those in large parts of Europe and North America as well as in Africaand India, is known as Helminthosporium turcicum or synonymously asExserohilum turcicum (Pass.) Leonard and Suggs (teleomorph: Setosphaeriaturcica (Luttrell) Leonard & Suggs). H. turcicum is the cause of theleaf spot disease known as “Northern Corn Leaf Blight” (NCLB), which canoccur in epidemic proportions during wet years, attacking vulnerablemaize varieties and causing a great deal of damage and considerablelosses of yield of 30% and more over wide areas (Perkins & Pedersen,1987; Raymundo & Hooker, 1981a; Ullstrup & Miles, 1957). Since the1970s, then, natural resistance in genetic material has been sought.Currently, quantitative and qualitative resistances are known. While theoligo- or polygenically inherited quantitative resistance appearsincomplete and non-specific as regards race in the phenotype and isinfluenced by additional and partially dominant genes, qualitativeresistance is typically race-specific and can be inherited throughindividual, mostly dominant genes such as Ht1, Ht2, Ht3, Htm1 or Htn1(Lipps et al., 1997; Welz & Geiger, 2000). Backcrosses in manyfrequently used inbred maize lines such as W22, A619, B37 or B73 havesuccessfully brought about introgression of the HT genes, where theyexhibit a partial dominance and expression as a function of therespective genetic background (Welz, 1998).

Despite this complex genetic architecture of NCLB resistance in maize,until now principally the use of the Ht1 gene in maize together with apartial quantitative resistance has been sufficient to controlhelminthrosporiosis (Welz, 1998). The basis for this is that globally,race 0 of H. turcicum dominates as regards use (approximately 55%)(Lipps et al., 1997; Ferguson & Carson, 2007), while other races such as2N and 23N are only rarely used and even then in a geographicallyrestricted area (Moghaddam & Pataky, 1994; Jordan et al., 1983; Lipps &Hite, 1982; Thakur et al., 1989; Welz, 1998). This race 0 is avirulenthaving regard to a maize plant with Ht1, so that when provided with asuitable quantitative resistance, it exhibits a sufficient generalresistance to NCLB. However, many studies have reported an increasingdissemination of the less common races (Jordan et al., 1983; Welz, 1998;Pratt & Gordon, 2006). The reasons for this are linked to the populationdynamic of a pathogen which allows changes in pathogen virulence by newmutations on avirulence genes and new combinations of availablevirulence genes. Finally, this can lead to the occurrence of new,suitable, sometimes more aggressive pathogenic races. In Brazil, forexample, the H. turcicum population already appears to be substantiallymore diverse having regard to the race composition than, for example, inNorth America. Gianasi et al. (1996) reported H. turcicum races whichhave already broken through the resistance conferred by the Ht1 gene. Inaddition, there is the instability of the resistance genes to certainenvironmental factors such as temperature and light intensity in someclimate zones (Thakur et al., 1989). This development has theconsequence that globally, the use of novel HT resistance genes or suchto which, until now, little attention has been paid for the productionof commercial maize plants is growing in importance in order to target abroader and more long-lasting resistance to H. turcicum in maize.Initial approaches in this regard were attempted as early as 1998 byPataky et al. The NCLB resistance in sh2 elite maize was improved byusing a combination of Ht1 and Htn1.

A source of monogenic Htn1 resistance is the Mexican landrace“Pepitilla” (Gevers, 1975). Htn1 introgression lines exhibit a genemapping on the long arm of chromosome 8 approximately 10 cM distal fromHt2 and 0.8 cM distal from the RFLP marker umc117 (bin 8.06) (Simcox &Bennetzen, 1993). In contrast to the usual HT resistance genes, Htn1confers resistance by delaying the onset of sporulation, and thuscombats the development of lesions. As a result, fewer, smaller lesionsas well as reduced sporulation zones are formed (Raymundo et al., 1981b,Simcox & Bennetzen, 1993). Chlorotic-necrotic lesions such as thosewhich occur with Ht1, Ht2 or Ht3-conferred resistance, are not formed(Gevers, 1975). However, the resistance reaction in the heterozygousstate of the Htn1 gene is significantly less effective than in thehomozygous state (Raymundo et al., 1981b).

The development of additional specific markers which could furthersimplify genotype determination would improve the breeding manageabilityof the Htn1 gene. Marker assisted selection (MAS) technology thus makesefficient stacking or pyramiding of several resistance genes possible(Min et al., 2012). The introgression lines B37Htn1 or W22Htn1 wereemployed in many studies on mapping the resistance locus and identifyingthe resistance source (Raymundo et al., 1981a, b; Simcox & Bennetzen,1993, Bar-Zur et al., 1998; Coates & White, 1998). Available informationregarding markers which could be used for selection of the resistancelocus for Htn1 from the accession Pepitilla, however, is still onlylimited (Simsox & Bennetzen, 1993). The known markers for Htn1 which arefunctional for and flank the resistance locus from the accessionPepitilla are still mapped at close to 22.2 cM apart, which in the bestcase scenario allows selection of a large chromosome fragment. However,there is a frequent risk that within this fragment between the markers,a double genetic recombination occurs which could result in a falsepositive selection for the Htn1 resistance locus. In addition, in somecases the probability of unwanted genetic regions being taken into theintrogression line rises with the size of the introgressed chromosomefragment and be transmitted over generations of elite lines. Suchgenetic regions, in particular when they are closely coupled with theHtn1 locus and lead to unequivocally negative effects on one or moreagronomic features, are known as linkage drag. From known studies whichinvestigated and used introgression lines with Htn1 from Pepitilla,however, such negative effects are unknown. Even the very comprehensiveresearch work by Welz (1998) which, inter alia, was also carried out onB37Htn1, postulated that in view of, for example, yield and ripening,introgression of the Htn1 locus brought about no significantdisadvantages. Thus, no serious efforts have been made in the prior artto deliberately shorten the large chromosome fragment.

In contrast, WO 2011/163590 discloses the genotype PH99N as analternative source for NCLB resistance on chromosome 8 bin 5 which,however, does not correspond to the accession Pepitilla. Essentially,only resistance as regards H. turcicum races 0 and 1 have beenidentified in backcross populations from PH99N. Even the resistancephenotype was not clearly determined. Nevertheless, the authorsconcluded that the resistance was due to the Htn1 gene. But theresistance locus in PH99N was restricted to only a ˜224 kb longchromosome fragment; a resistant maize plant with the 224 kb fragmentand thus the assumed Htn1 was not disclosed, however. In addition, thegenotype PH99N was not made available to the public by deposition.

An alternative approach to making the Htn1 gene useful is theidentification and cloning of the resistance gene and using it in atransgenic strategy.

With the intention of identifying the resistance gene for NCLB, in 2010,Chung et al. 2010 published a study for fine mapping the bin 8.06resistance locus. The chromosome fragment under investigation, however,was not derived from Pepitilla but from the maize hybrid DK888 whichexhibits multiple disease resistance. Investigations on Helminthosporiumrace specificity initially made it clear that the resistance locus onDK888, designated qNLB8.06_(DK888), was closely linked or functionallylinked with the Ht2 and Htn1 genes, since Helminthosporium strains 23and 23N were virulent (Chung et al., 2008). Positive detection of thepresence of Htn1 was not accomplished, however, in the absence of a pureN isolate from H. turcicum. In addition, the resistance phenotype withqNB8.06_(DK888) also did not correspond to the expected phenotype havingregard to the appearance of chlorotic lesions and the delay in lesionformation. Further detailed complementation studies in Chung et al.(2010) finally provided indications that qNLB8.06_(DK888) was eitheridentical to, allelic, closely linked or functionally linked with Ht2,but not with Htn1. The resistance locus qNLB8.06_(DK888) could beassigned to a chromosome fragment of 0.46 Mb. Genome annotations of thischromosome fragment hinted at 12 putative open reading frames, of whichthree could respectively be a tandem protein kinase-like gene(GRMZM2G135202; GRMZM2G164612) or a protein phosphatase-like gene(GRMZM2G119720) and each equally constituted promising candidate genesfor the resistance gene Ht2 (Chung et al., 2010). A functionalverification was not described.

Furthermore, WO 2011/163590 A1 also annotated the presumed Htn1 gene inthe resistance source PH99N as a tandem protein kinase-like gene(GRMZM2G451147) and disclosed its genetic sequence, but also did notdetermine its functionality, for example in a transgenic maize plant.

SUMMARY OF THE INVENTION

The present invention stems from the prior art described above; theobject of the present invention is to provide a maize plant whichexhibits resistance to the pathogen Helminthosporium turcicum from thedonor Pepitilla and wherein the agronomic features of known maize plantscan be overlaid with resistance from the donor Pepitilla.

The object is accomplished on the one hand by the provision of a maizeplant into the genome of which a chromosome fragment from the donorPepitilla has been integrated, wherein the chromosome fragment comprisesan interval of the donor (hereinafter termed the first interval orinterval 1) which exhibits donor alleles in accordance with thehaplotype shown in Table 2 and a polynucleotide which confers resistanceto Helminthosporium turcicum in the maize plant, and wherein thechromosome fragment does not contain a further interval of the donor(hereinafter termed the second interval or interval 2) between a markerin a first marker region (M1) which is flanked by the markers SYN14136and PZE-108076510 and a marker in a second marker region (M2) which isflanked by the markers SYN24931 and PZE-108077560. These and alternativesolutions to the problem, described below, may be based on a breedingprogramme for integration of the Htn1 locus from Pepitilla into maizelines. However, genetic engineering approaches may also be selected, bymeans of which plants in accordance with the present invention may beproduced. Examples of genetic engineering strategies are described inmore detail below. In order to produce the plants of the presentinvention, various genotypes from the prior art may be used. Inparticular, B37HTN1, which comprises the resistance locus for thelandrace “Pepitilla”, was used as the original line. In addition toPepitilla itself and B37HTN1 (also known in the prior art as B37HtN),almost any maize genotype may be called upon for integration of the Htn1locus in order to produce a maize plant in accordance with the inventioninto the genome of which, in particular on chromosome 8 bin 5 or 6, anintrogression of the Htn1 resistance locus from Pepitilla has beeninserted. In this respect, many examples of genotypes are known in theprior art, for example: W22Htn (e.g. Bar-Zur et al., 1998); H6314Htn(e.g. Bar-Zur et al.,1998), B73HtN (e.g. Shimoni et al., Journal ofPhytopathology 131:4 (1991), 315-321), B68HtN and A632HtN (e.g. Carson,Plant Disease 79 (1995), 717-720) and A619HtN (e.g. Stanković et al,Genetika 39:2 (2007), 227-240). In a maize plant in accordance with theinvention, the chromosome fragment derives from the donor Pepitilla; ina preferred embodiment of the maize plant in accordance with theinvention, the chromosome fragment derives from the donor B37HTN1 orfrom another maize genotype as cited above. As an example, B37HTN1 maybe ordered from the Maize Genetics COOP Stock Center using the stock ID65749.

The chromosome fragment integrated into the genome of the maize plant ofthe invention derives from the donor Pepitilla which, as is known,comprises the resistance locus HTN1. The introgression of thisresistance locus is localized on the long arm of chromosome 8, bin8.05-8.06. The integrated chromosome fragment comprises the firstinterval of the donor, which comprises a polynucleotide which confersresistance against Helminthosporium turcicum in the maize plant of theinvention. In this regard, the polynucleotide comprises one or moreresistance-conferring genes of the HTN1 locus from Pepitilla (Table 1)or gene alleles thereof. Under H. turcicum infestation conditions, thegene or gene allele may produce a resistance phenotype with featurestypical of HTN1. Preferably, the polynucleotide comprises one or moreresistance-conferring genes of the HTN1 locus, preferably fromPepitilla, selected from RLK1 and EXT1 (see Table 1) or gene allelesthereof which produce a resistance phenotype with the typical featuresof HTN1 under H. turcicum infestation conditions. Particularlypreferably, the polynucleotide comprises a nucleotide sequence whichcodes for a polypeptide in accordance with SEQ ID NO: 2 or SEQ ID NO: 6or a homologue of a polypeptide in accordance with SEQ ID NO: 2 or SEQID NO: 6, which produce a resistance phenotype with the typical featuresof HTN1 under H. turcicum infestation conditions. Examples of thesefeatures typical of HTN1 are delayed onset of sporulation, reduceddevelopment of lesions, development of smaller lesions, reducedsporulation zones and/or no or only isolated chlorotic-necrotic lesions.Structurally, the polynucleotide is characterized in that it comprises anucleic acid molecule which (a) comprises a nucleotide sequence inaccordance with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, (b) comprises anucleotide sequence with an identity of at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with one of the nucleotidesequences in accordance with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15,preferably over the entire length of the sequence, (c) which hybridizeswith the complementary strand of a nucleic acid molecule in accordancewith (a) or (b) under stringent conditions, (d) which codes for apolypeptide with an amino acid sequence in accordance with SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or 16, (e) which codes for a polypeptide with anamino acid sequence which has at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% A identity with one of the amino acidsequences in accordance with (d), or (f) which comprises a part sequenceof a nucleic acid in accordance with (a) to (e). In a preferredembodiment, the polynucleotide is characterized in that it comprises anucleic acid molecule which (aa) comprises a nucleotide sequence inaccordance with SEQ ID NO: 1 or 5, (bb) comprises a nucleotide sequencewith an identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% A with one of the nucleotide sequences inaccordance with SEQ ID NO: 1 or 5, preferably over the entire length ofthe sequence, (cc) which hybridizes with the complementary strand of anucleic acid molecule in accordance with (aa) or (bb) under stringentconditions, (dd) which codes for a polypeptide with an amino acidsequence in accordance with SEQ ID NO: 2 or 6, (ee) which codes for apolypeptide with an amino acid sequence which has at least 80%, aminoacid sequences in accordance with (dd), or (ff) which comprises a partsequence of a nucleic acid in accordance with (aa) to (ee). Theexpression “part sequence of a nucleic acid molecule” as used in thepresent invention may be at least 20, 30, 40, 50, 60, 70, 80, 90 or atleast 100 successive nucleotides, furthermore at least 150, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 successivenucleotides. The polynucleotide may be in the heterozygous or homozygousstate in the genome of the maize plant of the invention; preferably, thepolynucleotide is in the homozygous state.

TABLE 1 Potential resistance-conferring genes of the HTN1 locus fromPepitilla; Gene name (column 1); reference to corresponding SEQ ID Nosin the genomic exon sequence (column 2); reference to corresponding SEQID Nos in the predicted amino acid/protein sequence (column 3);annotated homologous gene from the B73 reference genome (column 4).Protein cDNA sequence Gene name SEQ ID NO: SEQ ID NO: Homologous B73gene RLK1 1 2 GRMZM2G451147 RLK4 3 4 GRMZM2G144028 EXT1 5 6GRMZM2G445338 DUF1 7 8 AC209075.3_FG007 ZNF1 9 10 GRMZM2G175661 CYT1 1112 GRMZM2G092018 RET1 13 14 GRMZM2G091973 HYD 15 16 GRMZM2G144021

Furthermore, the first interval in the chromosome fragment whichexhibits donor alleles in accordance with the haplotype in Table 2 ischaracterized by the sequence of donor alleles in the haplotype of Table2, but is not limited to this sequence of donor alleles in accordancewith Table 2. This means that the first interval exhibits at least thedonor allele which describes the resistance-conferring gene from Table1, optionally with the donor allele of the marker MA0008. Furthermore,the first interval preferably exhibits at least the donor alleles inaccordance with the haplotype of Table 2 from MA0021 to MA0022 (i.e.MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022) or fromMA0005 to MA0022 (i.e. MA0005, MA0021, MA0007, MA0008, MA0009, MA0010,MA0011, MA0012 and MA0022) or from MA0005 to MA0013 (i.e. MA0005,MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022 andMA0013) or from MA0005 to MA0014 (i.e. MA0005, MA0021, MA0007, MA0008,MA0009, MA0010, MA0011, MA0012, MA0022, MA0013 and MA0014) or fromMA0005 to MA0015 (i.e. MA0005, MA0021, MA0007, MA0008, MA0009, MA0010,MA0011, MA0012, MA0022, MA0013, MA0014 and MA0015) or from MA0005 toMA0016 (i.e. MA0005, MA0021, MA0007, MA0008, MA0009, MA0010, MA0011,MA0012, MA0022, MA0013, MA0014, MA0015 and MA0016), particularlypreferably from MA0005 to MA0017 (i.e. MA0005, MA0021, MA0007, MA0008,MA0009, MA0010, MA0011, MA0012, MA0022, MA0013, MA0014, MA0015, MA0016and MA0017), MA0005 to MA0018 (i.e. MA0005, MA0021, MA0007, MA0008,MA0009, MA0010, MA0011, MA0012, MA0022, MA0013, MA0014, MA0015, MA0016,MA0017 and MA0018), MA0005 to PZE-108095998 (i.e. MA0005, MA0021,MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022, MA0013, MA0014,MA0015, MA0016, MA0017, MA0018 and PZE-108095998), MA0005 toPZE-108096011 (i.e. MA0005, MA0021, MA0007, MA0008, MA0009, MA0010,MA0011, MA0012, MA0022, MA0013, MA0014, MA0015, MA0016, MA0017, MA0018,PZE-108095998 and PZE-108096011) or MA0005 to MA0019 (i.e. MA0005,MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022, MA0013,MA0014, MA0015, MA0016, MA0017, MA0018, PZE-108095998, PZE-108096011 andMA0019), more particularly preferably from MA0005 to PZE-108096610 (i.e.MA0005, MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022,MA0013, MA0014, MA0015, MA0016, MA0017, MA0018, PZE-108095998,PZE-108096011, MA0019 and PZE-108096610), MA0005 to MA0020 (i.e. MA0005,MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022, MA0013,MA0014, MA0015, MA0016, MA0017, MA0018, PZE-108095998, PZE-108096011,MA0019, PZE-108096610 and MA0020), MA0005 to PZE-108096791 (i.e. MA0005,MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012, MA0022, MA0013,MA0014, MA0015, MA0016, MA0017, MA0018, PZE-108095998, PZE-108096011,MA0019, PZE-108096610, MA0020 and PZE-108096791) or MA0005 to MA0006(i.e. MA0005, MA0021, MA0007, MA0008, MA0009, MA0010, MA0011, MA0012,MA0022, MA0013, MA0014, MA0015, MA0016, MA0017, MA0018, PZE-108095998,PZE-108096011, MA0019, PZE-108096610, MA0020, PZE-108096791 and MA0006).This resistant haplotype unequivocally specifies and identifies theresistance source Pepitilla. In particular, the first interval islocalized between the markers MA0004 and PZE-108097482, between themarkers MA0004 and MA0022, between the markers MA0005 and PZE-108097482or between the markers MA0005 and MA0022. Preferably, the first intervaldescribes a segment of the chromosome fragment which can confer theresistance typical of HTN1. As such it is a carrier of thepolynucleotide cited above.

TABLE 2 Resistant haplotype from B37HTN1; Position in bp on Allele donorB73 AGPv02 B37HTN1 Marker designation 151831049 C MA0005 151907173 GMA0021 152045106 T MA0007 152045141 T MA0008 152045402 T MA0009152045516 C MA0010 152045912 T MA0011 152046502 T MA0012 152046529 AMA0022 152133057 G MA0013 152133380 A MA0014 152144310 A MA0015152250992 A MA0016 152301656 A MA0017 152304127 A MA0018 152433358 APZE-108095998 152435855 A PZE-108096011 152630794 C MA0019 152703579 GPZE-108096610 152753635 A MA0020 152887338 G PZE-108096791 152888374 AMA0006

Furthermore, every maize plant in accordance with the invention is aHT-resistant maize plant. The HT resistance conferred by integration ofthe chromosome fragment may be quantified by determining classificationscores in phenotyping experiments in accordance with the scheme in Table3 and Example 1.A); in this, the resistance level reduces from 1 to 9.HT-resistant maize plants in accordance with the invention exhibit anincreased resistance to H. turcicum of at least 1 classification score,preferably at least 2 classification scores or at least 3 classificationscores and particularly preferably at least 4 classification scores.Preferably, a maize plant in accordance with the invention exhibitsresistance to at least one race of Helminthosporium turcicum which doesnot correspond to the known race specificity known in the prior art. Ina particularly preferred embodiment, a maize plant in accordance withthe invention is resistant to all known races of Helminthosporiumturcicum, i.e. the conferred resistance is not race-specific and may beparticularly advantageous in the formation of a broad resistance toHelminthosporium turcicum.

TABLE 3 Classification score scheme for phenotyping experiments in fieldtrials at various locations with natural and artificial H. turcicuminoculation (from the Deutsche Maiskomitee (DMK, German maizecommittee); AG variety 27.02.02; (DMK J. Rath; R P Freiburg H. J.Imgraben) Classification score Phenotype 1 Plants exhibit no symptoms ofdisease, 0% 2 Beginning of infestation, first small spots (less than 2cm) visible. Less than 5% of leaf surface affected. 3 Some spots havedeveloped on a leaf stage. Between 5-10% of leaf surface affected. 410-20% of leaf surface affected. Clearly visible spots on several leafstages. 5 20-40% of leaf surface affected. Spots start to coalesce. 640-60% of leaf surface affected. Systematic infestation visible onleaves. 7 60-80% of leaf surface affected. Approximately half of leavesdestroyed or dried out because of fungal infestation. 8 80-90% of leafsurface affected. More than half of leaves destroyed or dried outbecause of fungal infestation. 9 90-100% of leaf surface affected. Theplants are almost completely dried out.

The description discloses the genetic or molecular structure of the HTN1locus by providing a haplotype, by mapping prominent markers and also byidentifying candidate genes for conferring resistance to the pathogenHelminthosporium turcicum.

Surprisingly, the maize plants in accordance with the invention provedto be agronomic in phenotyping experiments carried out in the field andin the greenhouse. This is because, while other converted lines from abreeding programme for integration of the HTN1 locus from Pepitilla aswell as from known prior art converted lines such as B37HTN1, inaddition to the conferred HT resistance under non-infestation conditionswith H. turcicum and under comparable environmental conditions(temperature, nutrient supply, location etc) exhibited a significantdelay in the male and/or female flowering time compared with thecorresponding line without introgression (for example isogenic lines ororiginal lines), in the maize plant of the invention the flowering timecorresponded to that of a comparative isogenic maize plant into thegenome of which a chromosome fragment from the donor Pepitilla had notbeen integrated. The “flowering times” correspond when they differ fromeach other by less than 2 days. The magnitude of the observed delay inthis case is strongly dependent on the species of maize or the maizegenotype, the prevailing environmental conditions such as the soilcondition, humidity, precipitation, temperature etc and/or biotic stresssuch as pathogen infestation other than with H. turcicum. The delay wasat least 2 days, at least 3 days, at least 5 days or at least 7 days.This established difference in the flowering time is due to linkage dragas part of the introgression, which is particularly surprising sinceobservations of this type are not known in the prior art. The floweringtime is an important agronomic feature. It can directly andsubstantially influence the yield potential of a maize plant. A delayedflowering time usually results in a reduced yield.

In order to elucidate the genetic cause of this disadvantage and toidentify the linkage drag, extensive backcrossing programmes accompaniedby genotyping and phenotyping were carried out, for example. The workwas supported by intensive development of specific molecular markers onthe chromosome fragment carrying the HTN1. The techniques of markeraided selection (MAS) and carrying out focussed backcross programmes(for example “map based cloning”) can be found in the prior art (Gupta &Varshney, 2013). The QTL with HTN1 resistance from the donor B37HTN1 orPepitilla was localized with the aid of the SSR markers bnlg1067,umc1121, MA0002, MA0003, bnlg1782, umc1287, umc1960 and bnlg240 in thedescendants on chromosome 8 (bin 8.06) between the markers MA0002 (Table4) and umc1287 (Table 5) in a region of 23.1 cM (see FIG. 1). In maizeplants with the delayed flowering time, the locus of the genomic donorsequence segment which is responsible for the identified linkage drag ofthe flowering time was successfully determined to be on a further secondinterval of the donor on the chromosome fragment (Example 3B; FIG. 3).In a maize plant in accordance with the invention, a chromosome fragmentis integrated into it which does not contain the second interval of thedonor. Here, the second interval stems, for example, from a recurrentparent which does not carry the linkage drag for flowering time or froman exogenically introduced homologous DNA fragment which is not acarrier of the linkage drag, on a suitable donor vector for targetedhomologous recombination. The second interval is proximal and closelycoupled to the resistance locus HTN1 or to the first interval. Thesecond interval is an interval between a marker in a first marker region(M1) which is flanked by the markers SYN14136 and PZE-108076510 and amarker in a second marker region (M2) which is flanked by the markersSYN24931 and PZE-108077560. The flanking markers may be discerned fromTable 4. The markers SYN14136, PZE-108076510, SYN24931 and PZE-108077560are SNP markers for use in the KBioscience-KASP system(www.lgcgenomics.com/genotyping/KASP-genotyping-reagents/KASP-overview/).They clearly define the marker regions M1 and M2 either side of thesequence segment which in the donor B37HTN1 or Pepitilla carry thelinkage drag for flowering time. Moreover, as the polymorphic marker,these are also capable of differentiating between Pepitilla donoralleles and, for example, the allele for the recurrent parent. Alldetails regarding the use of these markers as a KASP marker can beobtained from Table 4. Suitable exemplary primer hybridizationparameters for the PCR are provided in Example 2. A person skilled inthe art is, moreover, also capable of determining other suitablehybridization parameters. Furthermore, it is routine for a personskilled in the art with a knowledge of the described marker regions inaddition to the cited markers to develop other markers, in particularpolymorphic markers, in M1 and/or M2. Using the markers cited here,namely SYN14136, PZE-108076510, SYN24931 and PZE-108077560 orself-developed markers in M1 and/or M2, the person skilled in the artwill readily be able to establish whether in a maize plant into thegenome of which a chromosome fragment with HTN1 resistance locus fromthe donor Pepitilla has been integrated, the second interval of thedonor described above is contained therein or not contained therein. Theperson skilled in the art will also be aware that, for example, duringthe course of a breeding process or a genetic engineering strategy fortargeted recombination, a chromosome interval can be removed from thedonor which, for example, comprises genomic sequences which causelinkage drag, by genetic/homologous recombination of the integratedchromosome fragment. In this regard, the interval of the Pepitilla donorcan be replaced by the corresponding interval of the recurrent parentgenome or by an exogenically introduced homologous DNA fragment. Markersin general and the markers disclosed here in particular can inparticular be used for selection in this regard. As an example, apossible use of markers for the detection of an allele will be givenbelow: detecting an allele may, for example, be carried out by (a)isolating at least one nucleic acid molecule from a genome of a plant ora plant cell/maize plant or maize plant cell, and (b) examining theisolated nucleic acid molecule with at least one marker, as well asoptionally (c) sequencing the allele in one and/or more genotypes, (d)detecting one and/or more polymorphisms and/or (e) restriction with arestriction endonuclease which can produce fragments of different sizesat a marker allele.

A preferred embodiment of the maize plant of the invention is a maizeplant as described above, wherein the chromosome fragment does notcontain the second interval of the donor which is flanked a) by themarkers SYN14136 and PZE-108077560, b) by the markers PZE-108076510 andPZE-108077560, c) by the markers SYN14136 and SYN24931 or d) by themarkers PZE-108076510 and SYN24931.

In a preferred embodiment, the maize plant of the invention exhibits adeviant male and/or female flowering time compared with thePepitilla-converted line or Pepitilla-converted plant such as B37HTN1which contains the interval 2 between a marker in a first marker region(M1) which is flanked by the markers SYN14136 and PZE-108076510, and bya marker in a second marker region (M2) which is flanked by the markersSYN24931 and PZE-108077560, wherein the term “deviant time” means thatthe converted line or converted plant exhibits a delay of at least 2days, at least 3 days, at least 5 days or at least 7 days.

A further preferred embodiment of the maize plant of the invention is amaize plant as described above, wherein the chromosome fragmentfurthermore does not contain an interval of the donor (hereinaftertermed the third interval or interval 3) between a marker in the secondmarker region M2 and a marker in a third marker region M3 which isflanked by the markers PZE-108093423 (Table 4) and PZE-108093748 (Table4). The markers PZE-108093423 and PZE-108093748 are SNP markers for usein the KBioscience-KASP-System(www.lgcgenomics.com/genotyping/KASP-genotyping-reagents/KASP-overview/).They unequivocally define the marker region M3. As polymorphic markers,they are also suitable for distinguishing between donor alleles and, forexample, the allele for the recurrent parent. All details regarding theuse of these markers as KASP markers can be obtained from Table 4.Suitable exemplary primer hybridization parameters for PCR are providedin Example 2. A person skilled in the art is also able to determineother suitable hybridization parameters. Furthermore, it is a routinematter for a person skilled in the art with a knowledge of the describedmarker region to develop other markers, in particular polymorphicmarkers, in M3 in addition to the cited markers. Using the markers forM2 as cited above and the markers PZE-108093423 and PZE-108093748 notedherein or self-developed markers in M3, it would be a simple matter fora person skilled in the art to establish whether, in a maize plant intothe genome of which a chromosome fragment with a HTN1 resistance locusfrom the donor Pepitilla has been integrated, contains or does notcontain the third interval of the donor as described above.

A further preferred embodiment of the maize plant in accordance with theinvention is provided by the maize plant as described above wherein thechromosome fragment does not contain a genetic segment which comprisesthe second interval and the third interval of the donor and is flankeda) by the markers SYN14136 and PZE-108093423, b) by the markersPZE-108076510 and PZE-108093423, c) by the markers SYN14136 andPZE-108093748 or d) by the markers PZE-108076510 and PZE-108093748.

In a further aspect, further genetic segments may be determined on thechromosome fragment which, under non infestation conditions with H.turcicum, could cause a significant negative influence on the yieldpotential of a maize plant into the genome of which a chromosomefragment with a HTN1 resistance locus from the donor Pepitilla has beenintegrated. Thus, independently of the delay to the flowering timedescribed above, converted lines as well as known prior art convertedlines such as B37HTN1, in addition to the conferred HT resistance,exhibit a substantially reduced yield, in particular a substantiallyreduced silage yield compared with the corresponding line withoutintrogression (for example isogenic line or original line). This is thecase even for lines into the genome of which a genetic segment of thedonor consisting of interval 2 (between a marker from M1 and M2) orinterval 2 and 3 (between a marker from M1 and M3) is no longer present.Observations of this type would not be expected by the person skilled inthe art, since there would be no indication in the prior art of alinkage drag of this type in HTN1 introgression lines. In order toelucidate the genetic cause of this agronomic disadvantage, for example,extended backcrossing programmes accompanied by genotyping andphenotyping were carried out. This work was supported by an intensivedevelopment of more accurate and more specific molecular markers on theHTN1-carrying chromosome fragment. In maize plants with the reducedyield (silage yield), the position of the genomic sequence segment whichis responsible for the linkage drag of the silage yield was successfullydetermined on two further intervals of the donor (hereinafter the fourthinterval or interval 4 and the fifth interval or interval 5) on thePepitilla chromosome fragment (Example 3C; FIG. 3). A maize plant inaccordance with the invention which comprises a corresponding intervalwithout linkage drag, for example from the recurrent parent, instead ofthe fourth and/or fifth interval of the donor carrying the linkage drag,exhibits no reduced silage yield, and thus a yield, in particular asilage yield, which is the same as or comparable to a line withoutintrogression (for example isogenic line or original line). Comparedwith a comparable maize plant with linkage drag for the silage yield,the silage yield of a maize plant in accordance with the inventionwithout fourth and/or fifth intervals of the donors, may be more than2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% higher. The fourthinterval is proximally located and closely coupled to the resistancelocus HTN1 or the first interval. The fifth interval is distally locatedand closely coupled with the resistance locus HTN1 or the firstinterval.

Thus, a particularly preferred embodiment of the maize plant of theinvention is a maize plant as described above wherein the chromosomefragment furthermore does not contain i) the fourth interval of thedonor between a marker in the third marker region M3 and a marker in afourth marker region M4 which is flanked by the markers MA0004 andMA0005, or ii) a genetic segment with the fourth interval between amarker in the third marker region M3 and a marker in a seventh markerregion M7 which is flanked by the markers MA0005 and MA0021, and/orwherein the chromosome fragment furthermore does not contain i) thefifth interval of the donor between a marker in a fifth marker region M5which is flanked by the markers MA0006 and PZE-108097482 and a marker ina sixth marker region M6 which is flanked by the markers PZE-108107671and SYN4196, or ii) a genetic segment with the fifth interval between amarker in an eighth marker region M8 which is flanked by the markersMA0022 and MA0013 and a marker in a sixth marker region M6 which isflanked by the markers PZE-108107671 and SYN4196. The flanking markersmay be obtained from Table 4. The markers MA0004, MA0005, MA0006,MA0013, MA0021, MA0022, PZE-108097482, PZE-108107671 and SYN4196 are SNPmarkers for use in the KBioscience-KASP system(www.lgcgenomics.com/genotyping/KASP-genotyping-reagents/KASP-overview/).They unequivocally define the marker regions M4, M5, M6, M7 and M8which, together with M3, establish the sequence segments which carry thelinkage drag for silage yield in the donor B37HTN1 or Pepitilla. Aspolymorphic markers, they are also suitable for distinguishing betweendonor alleles and, for example, the allele for the recurrent parent. Alldetails regarding the use of these markers as KASP markers can beobtained from Table 4. Suitable exemplary primer hybridizationparameters for PCR are provided in Example 2. A person skilled in theart is also able to determine other suitable hybridization parameters.

Furthermore, it is a routine matter for a person skilled in the art witha knowledge of the described marker region to develop other markers, inparticular polymorphic markers, in M4, in M5, in M6, in M7 and/or in M8.Using the markers MA0004, MA0005, MA0006, MA0013, MA0021, MA0022,PZE-108097482, PZE-108107671 and SYN4196 described here orself-developed markers in M4, in M5, in M6, in M7 and/or M8 togetherwith the markers in M3 described above, it would be a simple matter fora person skilled in the art to establish whether, in a maize plant intothe genome of which a chromosome fragment with a HTN1 resistance locusfrom the donor Pepitilla has been integrated, contains or does notcontain the fourth interval of the donor as described above.

A further particularly preferred embodiment of the maize plant of theinvention is a maize plant as described above wherein the chromosomefragment i) does not contain a genetic segment which comprises thesecond interval, the third interval and the fourth interval of the donorand is flanked a) by the markers SYN14136 and MA0004, b) by the markersPZE-108076510 and MA0004, c) by the markers SYN14136 and MA0005 or d) bythe markers PZE-108076510 and MA0005, or (ii) does not contain a geneticsegment which comprises the second interval and the third interval ofthe donor and is flanked a) by the markers SYN14136 and PZE-108093423,b) by the markers PZE-108076510 and PZE-108093423, c) by the markersSYN14136 and PZE-108093748 or d) by the markers PZE-108076510 andPZE-108093748, and the fifth interval of the donor, or (iii) does notcontain a genetic segment which comprises the second interval, the thirdinterval and the fourth interval of the donor and is flanked a) by themarkers SYN14136 and MA0004, b) by the markers PZE-108076510 and MA0004,c) by the markers SYN14136 and MA0005 or d) by the markers PZE-108076510and MA0005, and the fifth interval of the donor.

A further particularly preferred embodiment of the maize plant inaccordance with the invention is a maize plant as described above,wherein the chromosome fragment comprises (i) does not contain a geneticsegment which comprises the second interval, the third interval and thefourth interval of the donor and is flanked a) by the markers SYN14136and MA0021 or b) by the markers PZE-108076510 and MA0021, or (ii) doesnot contain a genetic segment which comprises the second interval, thethird interval and the fourth interval of the donor and is flanked a) bythe markers SYN14136 and MA0021 or b) by the markers PZE-108076510 andMA0021, and the fifth interval of the donor, or (iii) does not contain agenetic segment which comprises the second interval, the third intervaland the fourth interval of the donor and is flanked a) by the markersSYN14136 and MA0021 or b) by the markers PZE-108076510 and MA0021, and asecond genetic segment which comprises the fifth interval of the donorand is flanked a) by the markers MA0022 and PZE-108107671, b) by themarkers MA0022 and SYN4196, c) by the markers MA0013 and PZE-108107671or by the markers MA0013 and SYN4196, or (iv) does not contain a geneticsegment which comprises the second interval and the third interval ofthe donor and is flanked a) by the markers SYN14136 and PZE-108093423,b) by the markers PZE-108076510 and PZE-108093423, c) by the markersSYN14136 and PZE-108093748 or d) by the markers PZE-108076510 andPZE-108093748, and a second genetic segment which comprises the fifthinterval of the donor and is flanked a) by the markers MA0022 andPZE-108107671, b) by the markers MA0022 and SYN4196, c) by the markersMA0013 and PZE-108107671 or by the markers MA0013 and SYN4196, or (v)does not contain a genetic segment which comprises the second interval,the third interval and the fourth interval of the donor and is flankeda) by the markers SYN14136 and MA0021 or b) by the markers PZE-108076510and MA0021, and a second genetic segment which comprises the fifthinterval of the donor and is flanked a) by the markers MA0022 andPZE-108107671, b) by the markers MA0022 and SYN4196, c) by the markersMA0013 and PZE-108107671 or by the markers MA0013 and SYN4196.

The object forming the basis of the present invention is accomplished inan alternative manner by means of a maize plant into the genome of whicha chromosome fragment from the donor Pepitilla has been integrated,wherein the chromosome fragment comprises the first interval of thedonor which exhibits donor alleles in accordance with the haplotype ofTable 2 and comprises the polynucleotide which confers resistanceagainst Helminthosporium turcicum, and wherein the chromosome fragmentdoes not contain i) the fourth interval of the donor between a marker inthe third marker region which is flanked by the markers PZE-108093423and PZE-108093748, and a marker in the fourth marker region which isflanked by the markers MA0004 and MA0005, or ii) a genetic segment withthe fourth interval between a marker in the third marker region M3 and amarker in the seventh marker region M7 which is flanked by the markersMA0005 and MA0021. The above description, for example, as regardsmarkers the polynucleotide or the phenotyping is also valid in this caseand for every other alternative solution to the problem, as well asdisclosed embodiments.

A preferred embodiment of this inventive maize plant is a maize plant asdescribed above, wherein the chromosome fragment i) does not contain thefourth interval of the donor which is flanked a) by the markersPZE-108093423 and MA0004, b) by the markers PZE-108093748 and MA0004, c)by the markers PZE-108093423 and MA0005 or d) by the markersPZE-108093748 and MA0005, or ii) does not contain a genetic segmentwhich comprises the fourth interval of the donor and is flanked a) bythe markers PZE-108093423 and MA0021 or b) by the markers PZE-108093748and MA0021.

A further preferred embodiment of the maize plant in accordance with theinvention is a maize plant as hereinbefore described, wherein thechromosome fragment furthermore does not contain the third interval ofthe donor between a marker in the second marker region M2 and by amarker in the third marker region M3.

A further preferred embodiment of the maize plant in accordance with theinvention is a maize plant as described above, wherein the chromosomefragment does not contain a genetic segment which comprises the thirdinterval and the fourth interval of the donor and is flanked a) by themarkers SYN24931 and MA0004, b) by the markers PZE-108077560 and MA0004,c) by the markers SYN24931 and MA0005, d) by the markers PZE-108077560and MA0005, e) by the markers SYN24931 and MA0021 or f) by the markersPZE-108077560 and MA0021.

A further preferred embodiment of the maize plant in accordance with theinvention is a maize plant as hereinbefore described, wherein thechromosome fragment i) furthermore does not contain the fifth intervalof the donor between a marker in the fifth marker region M5 and a markerin the sixth marker region M6 or ii) does not contain a genetic segmentwith the fifth interval between a marker in the eighth marker region M8and a marker in the sixth marker region M6.

A further particularly preferred embodiment of the maize plant inaccordance with the invention is a maize plant as hereinbeforedescribed, wherein the chromosome fragment i) does not contain a geneticsegment which comprises the third interval and the fourth interval ofthe donor and is flanked a) by the markers SYN24931 and MA0004, b) bythe markers PZE-108077560 and MA0004, c) by the markers SYN24931 andMA0005 or d) by the markers PZE-108077560 and MA0005, and the fifthinterval, or ii) does not contain a genetic segment which comprises thethird interval and the fourth interval of the donor and is flanked a) bythe markers SYN24931 and MA0004, b) by the markers PZE-108077560 andMA0004, c) by the markers SYN24931 and MA0005 or d) by the markersPZE-108077560 and MA0005, and a second genetic segment which comprisesthe fifth interval and is flanked a) by the markers MA0022 and SYN4196,b) by the markers MA0022 and PZE-108107671, c) by the markers MA0013 andSYN4196 or by the markers MA0013 and PZE-108107671.

A further particularly preferred embodiment of the maize plant inaccordance with the invention is a maize plant as hereinbeforedescribed, wherein the chromosome fragment i) does not contain a geneticsegment which comprises the third interval and the fourth interval ofthe donor and is flanked a) by the markers SYN24931 and MA00021 or b) bythe markers PZE-108077560 and MA00021, and the fifth interval, or ii)does not contain a genetic segment which comprises the third intervaland the fourth interval of the donor and is flanked a) by the markersSYN24931 and MA00021 or b) by the markers PZE-108077560 and MA00021, anda second genetic segment which comprises the fifth interval and isflanked a) by the markers MA0022 and PZE-108107671, b) by the markersMA0022 and SYN4196, c) by the markers MA0013 and PZE-108107671 or by themarkers MA0013 and SYN4196.

Alternatively, the object of the present invention is furtheraccomplished by means of a maize plant, into the genome of which has achromosome fragment from the donor Pepitilla has been integrated,wherein the chromosome fragment comprises the first interval of thedonor which exhibits donor alleles in accordance with the haplotype ofTable 2 and which comprises the polynucleotide which confers resistanceagainst Helminthosporium turcicum in the maize plant, and wherein thechromosome fragment does not contain i) the fifth interval of the donorbetween a marker in the fifth marker region which is flanked by themarkers MA0006 and PZE-108097482, and a marker in the sixth markerregion which is flanked by the markers PZE-108107671 and SYN4196, or ii)a genetic segment with the fifth interval between a marker in the eighthmarker region M8 which is flanked by the markers MA0022 and MA0013, andby a marker in the sixth marker region M6 which is flanked by themarkers PZE-108107671 and SYN4196.

A further preferred embodiment of the maize plant in accordance with theinvention is a maize plant as hereinbefore described, wherein thechromosome fragment furthermore does not contain the third interval ofthe donor between a marker in the second marker region M2 and a markerin the third marker region M3.

A further particularly preferred embodiment of the maize plant inaccordance with the inventions is a maize plant as described above,wherein the chromosome fragment is flanked a) by a marker in the secondmarker region M2 and by a marker in the sixth marker region M6, b) by amarker in the third marker region M3 and by a marker in the sixth markerregion M6, c) by a marker in the fourth marker region M4 and by a markerin the sixth marker region M6, d) by a marker in the seventh markerregion M7 and by a marker in the sixth marker region M6, e) by a markerin the marker region M1 and by a marker in the marker region M5, f) by amarker in the second marker region M2 and by a marker in the fifthmarker region M5, g) by a marker in the third marker region M3 and by amarker in the fifth marker region M5, h) by a marker in the fourthmarker region M4 and by a marker in the fifth marker region M5, i) by amarker in the seventh marker region M7 and by a marker in the fifthmarker region M5, j) by a marker in the marker region M1 and by a markerin the marker region M8, k) by a marker in the second marker region M2and by a marker in the eighth marker region M8, l) by a marker in thethird marker region M3 and by a marker in the eighth marker region M8,m) by a marker in the fourth marker region M4 and by a marker in theeighth marker region M8, or n) by a marker in the seventh marker regionM7 and by a marker in the eighth marker region M8.

A further particularly preferred embodiment of the maize plant inaccordance with the invention is a maize plant as described above,wherein the chromosome fragment is flanked a) by the markers SYN24931and SYN4196, b) by the markers PZE-108077560 and SYN4196, c) by themarkers SYN24931 and PZE-108107671, d) by the markers PZE-108077560 andPZE-108107671, e) by the markers PZE-108093423 and SYN4196, by themarkers PZE-108093748 and SYN4196, g) by the markers PZE-108093423 andPZE-108107671, h) by the markers PZE-108093748 and PZE-108107671, i) bythe markers MA0004 and SYN4196, j) by the markers MA0005 and SYN4196, k)by the markers MA0004 and PZE-108107671, l) by the markers MA0005 andPZE-108107671, m) by the markers MA0021 and SYN4196, n) by the markersMA0021 and PZE-108107671, o) by the markers PZE-108076510 and MA0006, p)by the markers SYN14136 and MA0006, q) by the markers PZE-108076510 andPZE-108097482, r) by the markers SYN14136 and PZE-108097482, s) by themarkers SYN24931 and PZE-108097482, t) by the markers PZE-108077560 andPZE-108097482, u) by the markers SYN24931 and MA0006, v) by the markersPZE-108077560 and MA0006, w) by the markers PZE-108093423 andPZE-108097482, x) by the markers PZE-108093748 and PZE-108097482, y) bythe markers PZE-108093423 and MA0006, z) by the markers PZE-108093748and MA0006, aa) by the markers MA0004 and PZE-108097482, ab) by themarkers MA0005 and PZE-108097482, ac) by the markers MA0004 and MA0006,ad) by the markers MA0005 and MA0006, ae) by the markers MA0021 andPZE-108097482, af) by the markers MA0021 and MA0006, ag) by the markersPZE-108076510 and MA0013, ah) by the markers SYN14136 and MA0013, ai) bythe markers PZE-108076510 and MA0022, aj) by the markers SYN14136 andMA0022, ak) by the markers SYN24931 and MA0013, al) by the markersPZE-108077560 and MA0013, am) by the markers SYN24931 and MA0022, an) bythe markers PZE-108077560 and MA0022, ao) by the markers PZE-108093423and MA0013, ap) by the markers PZE-108093748 and MA0013, aq) by themarkers PZE-108093423 and MA0022, ar) by the markers PZE-108093748 andMA0022, as) by the markers MA0004 and MA0013, at) by the markers MA0005and MA0013, au) by the markers MA0004 and MA0022, av) by the markersMA0005 and MA0022, aw) by the markers MA0021 and MA0013, ax) by themarkers MA0021 and MA0022.

A further particularly preferred embodiment of the maize plant inaccordance with the inventions is a maize plant as described above,wherein the chromosome fragment is localized a) between a marker in thesecond marker region M2 and a marker in the sixth marker region M6, b)between a marker in the third marker region M3 and a marker in the sixthmarker region M6, c) between a marker in the fourth marker region M4 anda marker in the sixth marker region M6, d) between a marker in theseventh marker region M7 and a marker in the sixth marker region M6, e)between a marker in the first marker region M1 and a marker in the fifthmarker region M5 f) between a marker in the second marker region M2 anda marker in the fifth marker region M5, g) between a marker in the thirdmarker region M3 and a marker in the fifth marker region M5, h) betweena marker in the fourth marker region M4 and a marker in the fifth markerregion M5, i) between a marker in the seventh marker region M7 and amarker in the fifth marker region M5, j) between a marker in the markerregion M1 and a marker in the marker region M8, k) between a marker inthe second marker region M2 and a marker in the eighth marker region M8,l) between a marker in the third marker region M3 and a marker in theeighth marker region M8, m) between a marker in the fourth marker regionM4 and a marker in the eighth marker region M8, or n) between a markerin the seventh marker region M7 and a marker in the eighth marker regionM8.

TABLE 4 KASP marker primer sequences and assignment to B37HTN1 donoralleles derived from the landrace Pepitilla (allele X and allele Y:describe the biallelic values of the SNPs) Primer Primer Common Markeralleles alleles primer B37HTN1 position X (5′-3′) Y (5′-3′) (5′-3′)donor SNP AGPv02 [SEQ ID [SEQ ID [SEQ ID alleles Marker marker [bp] NO]NO] NO] (SNP) region SYN14136 131681497  17  18  19 A M1 PZE- 131905855 20  21  22 G M1 108076510 SYN24931 132877982  23  24  25 A M2 PZE-133189880  26  27  28 A M2 108077560 PZE- 150279048  29  30  31 A M3108093423 PZE- 150562764  32  33  34 G M3 108093748 PZE- 161543406  35 36  37 C M6 108107671 SYN4196 161766769  38  39  40 C M6 MA0004151688652  41  42  43 A M4 MA0005 151831049  44  45  46 C M4/M7 MA0021151907173 241 242 243 G M7 MA0006 152888310  47  48  49 A M5 PZE-153139646  50  51  52 A M5 108097482 MA0002 147720853  53  54  55 AMA0003 151346184  56  57  58 C MA0007 152045106  59  60  61 T MA0008152045141  62  63  64 T MA0009 152045402  65  66  67 T MA0010 152045516 68  69  70 C MA0011 152045912  71  72  73 T MA0012 152046502  74  75 76 A MA0022 152046529 244 245 246 A M8 MA0013 152133057  77  78  79 GM8 MA0014 152133380  80  81  82 T MA0015 152144310  83  84  85 A MA0016152250992  86  87  88 A MA0017 152301656  89  90  91 A MA0018 152304127 92  93  94 A MA0019 152630794  95  96  97 C MA0020 152753635  98  99100 A PZE- 152433358 101 102 103 T 108095998 PZE- 152435855 104 105 106A 108096011 PZE- 152703579 107 108 109 C 108096610 PZE- 152887338 110111 112 G 108096791

Furthermore, the present invention concerns a seed or grain, a tissue,an organ, a portion and a cell of the maize plants in accordance withthe invention described above. In this regard, the seed or the grain isa seed or a grain into the genome of which the chromosome fragment ofthe embodiment of the invention described above has been integrated.

In a further aspect, the present invention concerns a method foridentifying a H. turcicum-resistant maize plant into the genome of whicha chromosome fragment from the donor Pepitilla has been integrated,comprising the descendants of at least two alleles in the genome of theplant, wherein at least one allele is localized in a genomic segmentwhich is flanked by a marker in the first marker region M1, the secondmarker region M2, the third marker region M3, the fourth marker regionM4 or the seventh marker region M7, and by the polynucleotide describedabove which confers resistance to H. turcicum in the maize plant, andwherein at least one allele is localized in a genomic segment which isflanked by a marker in the sixth marker region M6, the fifth markerregion M5 or the eighth marker region M8. The marker regions andexemplary markers in these marker regions are described above.Preferably, the identified maize plant is a maize plant in accordancewith the invention. Furthermore, the invention also concerns a maizeplant which has been identified using the identification method whichhas been mentioned.

In a further aspect, the present invention concerns a method forincreasing the yield of a H. turcicum-resistant maize plant, into thegenome of which a chromosome fragment from the donor Pepitilla has beenintegrated, wherein the method comprises a step which removes the secondinterval of the donor, the fourth interval of the donor or the fifthinterval of the donor and wherein the chromosome fragment comprises thefirst interval of the donor described above which comprises donoralleles in accordance with the haplotype of Table 2 and a polynucleotidewhich confers resistance to Helminthosporium turcicum in the maizeplant. As an example, removal may be carried out by geneticrecombination during a crossing process between two maize plants,wherein a parent maize plant carries the HTN1-resistance locus fromPepitilla. In addition to the use of conventional breeding techniques toproduce a genetic recombination which has the result of replacing atleast one of the donor intervals with linkage drag identified above withgenomic sequences of the recurrent parent which are preferably free fromunwanted genes, modern biotechnology offers the person skilled in theart many tools which can enable precise genetic engineering to becarried out. Examples of known tools are meganucleases (Silva et al.,2011), homing endonucleases (Chevalier 2002), zinc finger nucleases,TALE nucleases (WO 2010/079430; WO 2011/072246) or CRISPR (Gaj et al.,2013). These are artificial nuclease fusion proteins which are capableof cleaving double stranded nucleic acid molecules such as plant DNA andthus of producing double strand breaks at desired positions in thegenome. By exploiting the cells own mechanisms for repairing induceddouble strand breaks, a homologous recombination or a “non-homologousend joining” can be carried out, which could lead to the removal of theintervals of the donor carrying linkage drag. Suitable target sequencesin the genome for the recognition domain nucleases may be taken, forexample, from the sequence information for the SNP markers (Table 4) orin their intervals. However, a person skilled in the art is also able toidentify other sequences, preferably within or between the six markerregions described above, which are suitable as target sequences for therecognition domains of the nucleases.

In this regard we shall now describe two genetic engineering approachesin more detail, with the aid of which the elimination of linkagedrag-carrying nucleotide sequences from a plant genome is supported ordirectly obtained. The following methods as well as the conventionalbreeding method may be employed for the production of the maize plantsin accordance with the invention.

As already stated, current molecular tools are capable of inducingdouble strand breaks at defined locations in the genome of a plant DNA.In this regard the use of TALE nucleases (TALENs) or zinc fingernucleases (ZFNs) has proved to be particularly advantageous. The TALE orZF recognition domain enables it to bind specifically to any location inthe genome. Knowing the sequence in the target region, the TALE or ZFrecognition domains can be tailored so that they exclusively bind todesired locations in the genome. If, for example, the recognitionsequence is fused with a non-specific endonuclease such as FokI, adouble strand break (DSB) can be induced at defined locations in thegenome, enabling targeted genome engineering (Tzfira et al., 2012; Li etal., 2011; Puchta and Hohn 2010). The person skilled in the art will befamiliar with handling FokI endonucleases and the provision of suitableTALENs and ZFNs from the prior art.

An induced double strand break may, for example, stimulate a homologousrecombination between an endogenic target gene locus (for example one ofthe above marker regions) and an exogenically introduced homologous DNAfragment which, for example, is not a carrier of linkage drag (forexample on a suitable donor vector). This so-called gene replacement orgenome editing can be carried out in vitro and does not necessitate anycrossing steps between two plants. To this end, the plants to bemodified must on the one hand be transiently transformed with nucleicacids coding for the designated TALENs or ZFNs, and on the other handwith the exogenic DNA fragment. The DNA fragment in this regard mayoriginate from a plant of the same species and, for example, correspondsto the chromosomal segment which is to be replaced, but without linkagedrag. After completing the induced homologous recombination, cells witha modified genome can be regenerated into plants and then selected as towhether the linkage drag has been successfully removed and thepreviously transformed DNA elements are once again lost during theregenerative cell division. The markers described above may also be usedfor this purpose. Methods for the transformation and regeneration areknown in the prior art and are also discussed further below.

Furthermore, the present TALENs and ZFNs may also be transgenicallyintroduced during the process of meiosis, where double strand breaks areinduced at predetermined locations in the genome and thus theprobability for a recombination at these locations in the crossing overstep is increased. In this manner, the elimination of linkage drag canbe significantly encouraged. A person skilled in the art is aware thatafter completion of meiosis, linkage drag-free and TALENs or ZFNs-freeplants are produced from the haploid cells. In a further aspect, thepresent invention concerns a method for the production of a maize plantin accordance with the invention, which comprises the following steps:(A) preparing a first maize plant into the genome of which a chromosomefragment from the donor Pepitilla has been integrated, wherein thechromosome fragment comprises a first interval of the donor whichexhibits donor alleles in accordance with the haplotype of Table 2 andcomprises a polynucleotide which confers resistance againstHelminthosporium turcicum in the maize plant, and wherein the chromosomefragment contains a second interval of the donor and/or the fourthinterval of the donor and/or the fifth interval of the donor, (B)providing a second maize plant, (C) crossing the maize plant from (A)with the maize plant from (B), and (D) selecting a maize plant inaccordance with the invention, preferably using at least one of themarkers described above. Alternatively, the present invention concerns amethod for the production of a maize plant in accordance with theinvention which comprises the following steps: (A) transientlytransforming a maize plant cell with a first nucleotide sequence whichcodes for a first protein with endonuclease activity (for example a TALEor ZF endonuclease fusion protein) which is capable of inducing a doublestrand break of the DNA between the marker regions M2 and M4 in themaize plant cell, and with a second nucleotide sequence which codes fora second protein with endonuclease activity (for example a TALE or ZFendonuclease fusion protein) which is capable of inducing a doublestrand break of the DNA in the genome of the maize plant cell betweenmarker regions M5 and M6, (B) transiently introducing a donor vectorinto the first maize plant cell which carries a chromosome fragment fromthe donor Pepitilla, wherein the chromosome fragment comprises a firstinterval of the donor which exhibits donor alleles in accordance withthe haplotype of Table 2 and comprises a polynucleotide which confersresistance against Helminthosporium turcicum in the maize plant, andwherein the chromosome fragment furthermore comprises the chromosomalsegments of the donor Pepitilla between the sites of the double strandbreak from (A) so that a homologous recombination takes place betweenthe genome of the first maize plant cell and the chromosome fragment ofthe donor vector, (C) regeneration of a maize plant from the maize plantcell, (D) identification of a maize plant in accordance with theinvention, preferably using at least one of the markers described above.Particularly preferably, transiently introduced first and second nucleicacid sequences and donor vectors are then lost. The person skilled inthe art will know how to carry this out and detect it.

In a further aspect, the invention encompasses the markers describedabove as oligonucleotides, in particular primer oligonucleotides.Preferably, the oligonucleotides are isolated oligonucleotides. Anoligonucleotide comprises a nucleic acid molecule with a nucleotidesequence selected from one of SEQ ID NOs: 41-49, 53-100 and 229-250.Furthermore, the present invention concerns the use of anoligonucleotide which comprises a nucleic acid molecule with anucleotide sequence selected from one of the SEQ ID NOs: 17-250, for theidentification of a H. turcicum-resistant maize plant. Preferably, theresistance derives from the donor Pepitilla and is HTN1.

Furthermore, the problem of the present invention is alternativelysolved by means of a transgenic plant, in particular a transgenic maizeplant, which comprises a transgenic plant cell as described below.Furthermore, the invention also concerns a portion of this plant inaccordance with the invention, wherein a portion may be a cell, atissue, an organ or a fusion of several cells, tissues or organs. Anexample of a fusion of several organs is a flower or a seed. In aparticular embodiment, the invention concerns a seed from the transgenicplant, wherein the seed comprises the polynucleotide in accordance withthe invention as the transgene, as described below. Preferably, atransgenic plant in accordance with the present invention, in particulara plant of the species Zea mays, exhibits a higher resistance to H.turcicum than a corresponding non-transformed plant (isogenic plantwithout the transgene). A transgenic HT-resistant plant in accordancewith the invention exhibits an increased resistance to H. turcicum of atleast one classification score, preferably at least 2 classificationscores or at least 3 classification scores and particularly preferablyat least 4 classification scores (see classification score scheme inTable 3).

Furthermore, the invention provides a method for the production of atransgenic plant which comprises a step for introducing thepolynucleotide of the invention or the vector of the present inventiondescribed below into a plant cell, and optionally a step for selectionof a transgenic plant cell. Furthermore, a method of this type for theproduction of a transgenic plant is characterized by a subsequent stepwhich includes the regeneration of the transgenic plant from thetransgenic plant cell produced in the first step. Methods forregeneration are known to the person skilled in the art from the priorart.

In an additional aspect, the present invention discloses thepolynucleotide which contains one or more resistance-conferring genes ofthe HTN1 locus from Pepitilla (table 1) or selected from RLK1 and EXT1(see Table 1) or gene alleles thereof. Genes or gene alleles may bringabout a resistance phenotype with the features typical of HTN1 underinfestation conditions with H. turcicum. Structurally, thepolynucleotide is characterized in that it comprises a nucleic acidmolecule that (a) comprises a nucleotide sequence in accordance with SEQID NO: 1, 3, 5, 7, 9, 11, 13 or 15, (b) comprises a nucleotide sequencewith an identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% A with one of the nucleotide sequences inaccordance with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15, preferably overthe entire length of the sequence, (c) which hybridizes with thecomplementary strand of a nucleic acid molecule in accordance with (a)or (b) under stringent conditions, (d) which codes for a polypeptidewith an amino acid sequence in accordance with SEQ ID NO: 2, 4, 6, 8,10, 12, 14 or 16, or (e) which codes for a polypeptide with an aminoacid sequence which has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% A identity with one of the amino acid sequences inaccordance with (d). In a preferred embodiment, the polynucleotide ischaracterized in that it comprises a nucleic acid molecule which (aa)comprises a nucleotide sequence in accordance with SEQ ID NO: 1 or 5,(bb) comprises a nucleotide sequence with an identity of at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% A with one ofthe nucleotide sequences in accordance with SEQ ID NO: 1 or 5,preferably over the entire length of the sequence, (cc) which hybridizeswith the complementary strand of a nucleic acid molecule in accordancewith (aa) or (bb) under stringent conditions, (dd) which codes for apolypeptide with an amino acid sequence in accordance with SEQ ID NO: 2or 6, or (ee) which codes for a polypeptide with an amino acid sequencewhich has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% A identity with one of the amino acid sequences in accordancewith (dd). Preferably, the polynucleotide can be isolated and/orpurified from its natural genetic environment or is present essentiallyin the pure or homogeneous form. Preferably, the polynucleotide is DNA,and particularly preferably cDNA, i.e. the polynucleotide comprises thecDNA from one or more resistance-conferring genes (Table 1). However, itmay also be present as RNA. The person skilled in the art will know howto deduce the genomic DNA sequence from the sequence informationdisclosed herein. A polynucleotide in accordance with the inventioncodes for at least one polypeptide which is capable of conferring aresistance against the pathogen Helminthosporium turcicum in a plant inwhich the polypeptide Is expressed. Preferably, the polypeptide which iscoded by the polynucleotide of the invention or portions thereof,preferably confers resistance to the pathogen Helminthosporium turcicum,in particular in a plant of the genus Zea or in a plant of the speciesZea mays.

Furthermore, the present invention also concerns a polypeptide which iscapable of conferring resistance to H. turcicum in a plant in which thepolypeptide is expressed and which is encoded by the polynucleotide ofthe invention or a portion thereof. Preferably, the polypeptidecomprises an amino acid sequence in accordance with SEQ ID NO: 2, 4, 6,8, 10, 12, 14 or 16 or, particularly preferably, an amino acid sequencein accordance with SEQ ID NO: 2 or 6. The polypeptide may be an isolatedpolypeptide.

In a further aspect, the present invention concerns a vector whichcomprises the polynucleotide in accordance with the invention. Thevector may be a plasmid, a cosmid, a phage or an expression vector, atransformation vector, shuttle vector or cloning vector, it may bedouble or single stranded, linear or circular, or it may be aprokaryotic or eukaryotic host, either by integration into its genome ortransforming extrachromosomally. Preferably, the polynucleotide of theinvention is operatively linked in an expression vector with one or moreregulatory sequences which allow transcription and optionally expressionin a prokaryotic or eukaryotic host cell. As an example, thepolynucleotide may be under the control of suitable promoters or aterminator. Suitable promoters may be promoters which are constitutivelyinduced (example: 35S promoter from the “cauliflower mosaic virus”(Odell et al. 1985); particularly suitable promoters are those promoterswhich are pathogen-inducible (example: PR1 promoter from parsley(Rushton et al., 1996)). Particularly suitable pathogen-induciblepromoters are synthetic or chimeric promoters which do not occur innature, are composed of several elements and contain a minimum promoteras well as, upstream of the minimum promoter, at least onecis-regulatory element which act as the binding site for specialtranscription factors. Chimeric promoters are custom-designed and areinduced by various factors or re-primed. Examples of such promoters canbe found in WO 2000/29592 and WO 2007/147395. An example of a suitableterminator is the nos-terminator (Depicker et al., 1982).

In addition to the vectors described above, the present invention alsoprovides a method which comprises introducing a vector as described intoa host cell. The vector may, for example, be introduced by conjugation,mobilization, biolistic transformation, agrobacterium-conferredtransformation, transfection, transduction, vacuum infiltration orelectroporation. Methods of this type as well as methods for thepreparation of the vectors described are familiar to the person skilledin the art (Sambrook et al. 2001).

In a further aspect, the present invention concerns a host cell whichcomprises the polynucleotide of the invention or a vector of the presentinvention. In the context of the invention, a host cell may be aprokaryotic cell (for example bacterial) or eukaryotic cell (for examplea plant cell or a yeast cell). Preferably, the enzyme is anagrobacterium such as Agrobacterium tumefaciens or Agrobacteriumrhizogenes, or a plant cell which comprises the polynucleotide of theinvention or the vector of the present invention. The person skilled inthe art will be familiar with the many methods such as conjugation orelectroporation for introducing the polynucleotide of the invention orthe vector of the present invention into an agrobacterium, and alsomethods such as various transformation methods (biolistictransformation, agrobacterium-conferred transformation) with which thepolynucleotide of the invention or the vector of the present inventioncan be introduced into a plant cell (Sambrook et al. 2001).

In a further aspect, the present invention concerns a transgenic plantcell which comprises the polynucleotide in accordance with the inventionas a transgene or the vector of the present invention. A transgenicplant cell of this type is, for example, a plant cell which istransformed with the polynucleotide in accordance with the invention orwith the vector of the present invention, preferably in a stable manner.In a preferred embodiment of the transgenic plant cell, thepolynucleotide is operatively linked with one or more regulatorysequences which allow transcription and optionally expression in theplant cell. The total construct of the polynucleotide in accordance withthe invention and the regulatory sequence(s) may then constitute thetransgene. Examples of regulatory sequences of this type are a promoteror a terminator. The person skilled in the art will be familiar withmany functional promoters and terminators which can be used in plants.Preferably, a transgenic plant cell in accordance with the presentinvention, in particular a cell of a plant of the species Zea mays,exhibits a higher resistance to H. turcicum than a correspondingnon-transformed plant cell (the (isogenic) plant cell without thetransgene). Transgenic HT-resistant plant cells of the invention exhibitan increased resistance to H. turcicum by at least one classificationscore, preferably at least 2 classification scores or at least 3classification scores and particularly preferably at least 4classification scores (see the classification scheme in Table 3).Furthermore, the present invention also concerns a method for theproduction of a transgenic plant cell of the present invention, whichcomprises a step for introducing the polynucleotide in accordance withthe invention or the vector of the present invention into a plant cell.As an example, the introduction may be carried out by transformation,preferably by stable transformation. Suitable techniques forintroduction such as biolistic transformation, agrobacterium-conferredtransformation or electroporation are known to the person skilled in the(Sambrook et al. 2001).

In a further aspect, the present invention also concerns a method forconferring or increasing a resistance to H. turcicum in a plant,preferably a plant of the species Zea mays, which comprises a step fortransformation of a plant cell with a polynucleotide in accordance withthe invention or with the vector of the present invention. Preferably,this method results in enhanced resistance to H. turcicum by at least 1classification score, preferably at least 2 classification scores or atleast 3 classification scores and particularly preferably at least 4classification scores (see the classification scheme in Table 3).

In an additional aspect, the present invention also encompasses a methodfor modification of the resistance phenotype of a plant, in particular amaize plant, to the pathogen Helminthosporium turcicum, which comprisesa step for mutation of the resistance-conferring gene of the HTN1 locusfrom Pepitilla or a gene allele comprised therein. Preferably, theresistance-conferring gene of the HTN1 locus from Pepitilla codes for apolypeptide in accordance with SEQ ID NO: 2 or a homologue of apolypeptide in accordance with SEQ ID NO: 2 which produces a resistancephenotype with the features typical of HTN1 under infestation conditionswith H. turcicum. The resistance-conferring gene of the HTN1 locus fromPepitilla or a gene allele thereof can be transgenic or endogenic in thegenome of the plant. Modification of the resistance phenotype can mean achange in the pathogen race specificity and/or a change in theresistance level, measured as the classification score based on thephenotypical characteristics such as the affected leaf surface (seeTable 3) or measured as an AUDPC value (see Example 1.C). Preferably,the resistance level after modification of the resistance phenotype isbetween the resistance level of a plant which expresses the non-mutatedresistance-conferring gene of the HTN1 locus from Pepitilla and theresistance level of an isogenic plant which does not express theresistance-conferring gene of the HTN1 locus from Pepitilla; however, itmay also be above the resistance level of a plant which expresses thenon-mutated resistance-conferring gene of the HTN1 locus from Pepitilla.Particularly preferably, the resistance level is between the resistancelevel of a plant which expresses the polypeptide in accordance with SEQID NO: 2 and the resistance level of an isogenic plant which does notexpress the polypeptide in accordance with SEQ ID NO: 2; it may also,however, be above the resistance level of a plant which expresses thepolypeptide in accordance with SEQ ID NO: 2. The expression “mutate” asused herein may be a change carried out by man in the genetic sequence(mutation). Examples in this regard are that plants, plant cells orplant portions receiving a high dose of chemical, radiological or othermutating agents and then selecting for mutants. Alternatively, themutation may also be carried out with, for example, the help of TILLINGnucleases, TALE nucleases, zinc finger nucleases or a CRISPR/Cas system,or by fusion, insertion, deletion or exchange in the DNA sequence or theamino acid sequence. The person skilled in the art may receivesufficient technical instruction from the prior art regarding carryingout the mutation steps. Preferably, mutation of theresistance-conferring gene of the HTN1 locus from Pepitilla results inat least one amino acid exchange, at least two amino acid exchanges, atleast three amino acid exchanges, or at least five or more amino acidexchanges. In the case of a plurality of amino acid exchanges, they maybe carried out on different gene alleles for the resistance-conferringgene of the HTN1 locus from Pepitilla, i.e. the mutation may beheterozygous or it may also be homozygous.

In a preferred embodiment of the method for the modification of theresistance phenotype of a plant, mutation of the resistance-conferringgene of the HTN1 locus from Pepitilla results in a point mutation in thenucleotide sequence in accordance with SEQ ID NO: 1 at position 1365with base exchange of a G for an A or at position 1490 with baseexchange of a G for an A. Furthermore, this embodiment also concerns amutation which leads to an amino acid exchange in the amino acidsequence in accordance with SEQ ID NO: 2 at position 455 from M(methionine) to I (isoleucine) or at position 497 from G (glycine) to E(glutamic acid). In a further preferred embodiment of the method,mutation of the resistance-conferring gene of the HTN1 locus fromPepitilla results in a point mutation, which results in an amino acidexchange in the nucleotide sequence in accordance with SEQ ID NO: 1between position 1365 and position 1490, or the embodiment concerns themutation which leads to an amino acid exchange in the amino acidsequence in accordance with SEQ ID NO: 2 between position 455 andposition 497.

In a further aspect, the invention concerns a method for producing aplant, in particular a maize plant, having a modified resistancephenotype as regards the pathogen Helminthosporium turcicum, whichcomprises a step for mutation of the resistance-conferring gene of theHTN1 locus from Pepitilla or a gene allele thereof in at least one cellof the plant or in at least one cell from which the plant isregenerated. Furthermore, the method can thus comprise a step forregeneration of at least one plant from at least one mutated cell andselection of the regenerated plants on the basis of the modifiedresistance phenotype as regards the pathogen Helminthosporium turcicum.Preferably, the resistance-conferring gene of the HTN1 locus fromPepitilla codes for a polypeptide in accordance with SEQ ID NO: 2 or ahomologue of a polypeptide in accordance with SEQ ID NO: 2, whichproduces a resistance phenotype with the features typical of HTN1 underinfestation conditions with H. turcicum. The resistance-conferring geneof the HTN1 locus from Pepitilla or a gene allele thereof may be presentin the plant transgenically or endogenically. Modification of theresistance phenotype can mean a change in the pathogen race specificityand/or a change in the resistance level, measured as the classificationscore based on the phenotypical characteristics such as the affectedleaf surface (see Table 3) or measured as an AUDPC value (see Example1.C). Preferably, the resistance level of the modified resistancephenotype lies between the resistance level of a plant which expressesthe non-mutated resistance conferred gene of the HTN1 locus fromPepitilla and the resistance level of an isogenic plant which does notexpress the resistance conferred gene of the HTN1 locus from Pepitilla;however, it may be above the resistance level of a plant which expressesthe non-mutated resistance conferred gene of the HTN1 locus fromPepitilla. Particularly preferably, the resistance level is between theresistance level of a plant which expresses the polypeptide inaccordance with SEQ ID NO: 2 and the resistance level of an isogenicplant which does not express the polypeptide in accordance with SEQ IDNO: 2; however, it can also be above the resistance level of a plantwhich expresses the polypeptide in accordance with SEQ ID NO: 2. Theexpression “mutation” herein may be understood to be a change in thegenetic sequence (mutation) carried out by man. Examples in this regardare plants, plant cells or plant parts receiving a high dose ofchemical, radiological or other mutagens and then being selected formutants. Alternatively, mutation may also be carried out, for example,with the aid of TILLING nucleases, TALE nucleases, zinc finger nucleasesor a CRISPR/Cas system or by fusion, insertion, deletion or exchanges inthe DNA sequence or the amino acid sequence. The person skilled in theart may receive sufficient technical instruction from the prior artregarding carrying out the mutation steps. Preferably, mutation of theresistance-conferring gene of the HTN1 locus from Pepitilla results inat least one amino acid exchange, at least two amino acid exchanges, atleast three amino acid exchanges, at least five or in more amino acidexchanges. In the case of a plurality of amino acid exchanges, these mayalso be present on different gene alleles of the resistance-conferringgene of the HTN1 locus from Pepitilla, i.e. the mutation may beheterozygous or even homozygous.

In a preferred embodiment of a method for the production of a planthaving a modified resistance phenotype as regards the pathogenHelminthosporium turcicum, mutation of the resistance-conferring gene ofthe HTN1 locus from Pepitilla results in a point mutation in thenucleotide sequence in accordance with SEQ ID NO: 1 at position 1365with base exchange of a G for an A or at position 1490 with baseexchange of a G for an A.

Furthermore, this embodiment also concerns a mutation which leads to anamino acid exchange in the amino acid sequence in accordance with SEQ IDNO: 2 at position 455 from M (methionine) to I (isoleucine) or atposition 497 from G (glycine) to E (glutamic acid). In a furtherpreferred embodiment of the method, mutation of theresistance-conferring gene of the HTN1 locus from Pepitilla results in apoint mutation, which results in an amino acid exchange in thenucleotide sequence in accordance with SEQ ID NO: 1 between position1365 and position 1490, or the embodiment concerns the mutation whichleads to an amino acid exchange in the amino acid sequence in accordancewith SEQ ID NO: 2 between position 455 and position 497.

The invention also concerns plants or parts thereof which may beproduced by a method for the production of a plant with a modifiedresistance phenotype as regards the pathogen Helminthosporium turcicum.

Further, the invention encompasses a plant or a part thereof whichcomprises a mutation in the resistance-conferring gene of the HTN1 locusfrom Pepitilla or a gene allele thereof. Preferably, the mutationresults in a modified resistance phenotype as described above.Preferably, the resistance-conferring gene of the HTN1 locus fromPepitilla codes for a polypeptide in accordance with SEQ ID NO: 2 or ahomologue of a polypeptide in accordance with SEQ ID NO: 2, whichproduces a resistance phenotype with the features typical of HTN1 underinfestation conditions with H. turcicum. The resistance-conferring geneof the HTN1 locus from Pepitilla or a gene allele thereof may be presentin the plant transgenically or endogenically. In a preferred embodiment,of the plant or the part thereof, the mutation is a point mutation inthe nucleotide sequence in accordance with SEQ ID NO: 1 at position 1365with base exchange of a G for an A or at position 1490 with baseexchange of a G for an A. Furthermore, this embodiment also concerns amutation which leads to an amino acid exchange in the amino acidsequence in accordance with SEQ ID NO: 2 at position 455 from M(methionine) to I (isoleucine) or at position 497 from G (glycine) to E(glutamic acid). In a further preferred embodiment of the plant or thepart thereof, the mutation of the resistance-conferring gene of the HTN1locus from Pepitilla is a point mutation which results in an amino acidexchange in the nucleotide sequence in accordance with SEQ ID NO: 1between the position 1365 and the position 1490, or the embodimentconcerns a mutation which leads to an amino acid exchange in the aminoacid sequence in accordance with SEQ ID NO: 2 between position 455 andposition 497.

Some of the terms used in this application will now be explained in moredetail:

The term “allele” refers to one or two or more nucleotide sequences at aspecific locus in the genome. A first allele is on a chromosome, asecond on a second chromosome at the same position. If the two allelesare different, they are heterozygous, and if they are the same, they arehomozygous. Various alleles of a gene (gene alleles) differ in at leastone SNP. Depending on the context of the description, an allele alsomeans a single SNP which, for example, allows for a distinction betweenthe donor of HTN1 (Pepitilla) and recurrent parent.

The expression “chromosome fragment” means a specific chromosomal DNAsegment of a specific chromosome which comprises at least one gene. Anintegrated chromosome fragment derives from a donor source. In thecontext of the invention, the sequential succession of the genes withinan integrated chromosome fragment corresponds to that sequence as it ispresent in the original chromosome fragment of the donor source. In thismanner, the integrated chromosome fragment may be present over the wholelength unchanged compared with the corresponding chromosome fragment inthe donor source. A chromosome fragment or a part thereof may constitutea specific “haplotype”, wherein the chromosome fragment may comprisespecific SNPs through which the haplotype can also be unequivocallyspecified and identified.

The terms “distal” and “proximal” describe the position of a chromosomalinterval or a genetic segment in relation to a specific reference point(for example a specific polynucleotide, another chromosomal interval ora gene) on a whole chromosome; “distal” means that the interval or thesegment is localized on the side of the reference point distant from thechromosome centromere, and “proximal” means that the interval or thesegment is localized on the side of the reference point close to thechromosome centromere.

“close coupled” or “closely linked” means two loci, two intervals, twogenetic segments or two markers (marker loci) which are less than 15 cM,less than 12 cM, less than 10 cM, less than 8 cM, less than 7 cM, lessthan 6 cM, less than 5 cM, less than 4 cM, less than 3 cM, less than 2cM, less than 1 cM, less than 0.5 cM, less than 0.2 cM, less than 0.1 cMdistant from each other, established using the IBM2 neighbors 4 geneticmap which is publicly available on the Maize GDB website.

The term “yield” as used in the context of the present invention refersto the productivity per unit area of a specific plant product withcommercial value. As an example, the yield of maize is usually measuredin metric tonnes of seed or grain per hectare (ha) and season or inmetric tonnes of dry biomass per hectare (ha) and season. Unlessotherwise specifically stated or specified, the yield may mean theabsolute fresh or dry matter, the relative fresh or dry matter, thesilage yield (also known as the silo maize yield or total dry matteryield) or the grain yield. The yield is influenced by genetic andenvironmental factors and in principle is a combination of manyagronomic properties which are built up of features based on geneticelements of a plant and contribute to the final yield during the season.Examples of these individual agronomic properties are seed emergence,vegetative vitality, stress tolerance, disease resistance or tolerance,herbicide resistance, branching tendency, flowering time, seed clusters,seed density, stability and storeability, threshing capability (uniformripening), etc.

The expression “genetic segment with” a more precisely specifiedinterval should be understood to mean a genetic segment which enclosesor comprises the more precisely specified interval, i.e. is not limitedto the more precisely specified interval. As an example, a “geneticsegment with the fifth interval between a marker in the eighth markerregion M8 which is flanked by the markers MA0022 and MA0013, and amarker in the sixth marker region M6 which is flanked by the markersPZE-108107671 and SYN4196” means that the genetic segment comprises thefifth interval and the genetic segment are localized between a marker inthe eighth marker region M8 which is flanked by the markers MA0022 andMA0013 and a marker in the sixth marker region M6 which is flanked bythe markers PZE-108107671 and SYN4196.

The term “hybridize” or “hybridization” should be understood to mean aprocedure in which a single stranded nucleic acid molecule agglomerateswith a nucleic acid strand which is as complementary as possible, i.e.base-pairs with it. Examples of standard methods for hybridization havebeen described in 2001 by Sambrook et al. Preferably, this should beunderstood to mean that at least 60%, more preferably at least 65%, 70%,75%, 80% or 85%, particularly preferably 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the bases of the nucleic acid molecule undergobase pairing with the nucleic acid strand which is as complementary aspossible. The possibility of such agglomeration depends on thestringency of the hybridization conditions. The term “stringency” refersto the hybridization conditions. High stringency is when base pairing ismore difficult, low stringency is when base pairing is easier. Thestringency of the hybridization conditions depends, for example, on thesalt concentration or ionic strength and the temperature. In general,the stringency can be increased by raising the temperature and/or byreducing the salt content. The term “stringent hybridization conditions”should be understood to mean those conditions under which ahybridization takes place primarily only between homologous nucleic acidmolecules. The term “hybridization conditions” in this respect refersnot only to the actual conditions prevailing during actual agglomerationof the nucleic acids, but also to the conditions prevailing during thesubsequent washing steps. Examples of stringent hybridization conditionsare conditions under which primarily only those nucleic acid moleculesthat have at least 70%, preferably at least 75%, at least 80%, at least85%, at least 90% or at least 95% sequence identity undergohybridization. Stringent hybridization conditions are, for example:4×SSC at 65° C. and subsequent multiple washes in 0.1×SSC at 65° C. forapproximately 1 hour. The term “stringent hybridization conditions” asused herein may also mean: hybridization at 68° C. in 0.25 M sodiumphosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours andsubsequently washing twice with 2×SSC and 0.1% SDS at 68° C. Preferably,hybridization takes place under stringent conditions.

The term “interval” or “chromosomal interval” means a continuous linearsegment on a genomic DNA which is present in an individual chromosome ina plant or on a chromosome fragment and which is usually defined throughtwo markers which represent the end points of the interval on the distaland proximal side. In this regard, the markers which define the ends ofthe interval may themselves also be a part of the interval. Furthermore,two different intervals might overlap. In the description, an intervalis specified by the statement “between marker A and marker B”. An endmarker of an interval may also be localized in a defined marker regionto one side of the interval. A marker region is then defined byproviding two flanking markers and constitutes a chromosomal segment onwhich more markers might be located, in addition to the flankingmarkers. Flanking markers determine the end points of a marker regionand are themselves still a part of the marker region. If both endmarkers of an interval are markers in different marker regions on bothsides of an interval, the description specifies an interval by stating“between a marker in a marker region X which is flanked by the markers Cand D and a marker in a marker region Y which is flanked by markers Eand F”. A marker region may extend over up to 500 000 base pairs (bp),and can preferably be between 100 000 and 400 000 bp in size, or canparticularly preferably be between 140 000 and 315 000 bp in size.

The term “introgression” as used in connection with the presentinvention means the transfer of at least one desired gene allele on agenetic locus of a genetic background into another. As an example, anintrogression of a desired gene allele at a specific locus may betransferred to a descendant by sexual crossing between two parents ofthe same species. Alternatively, for example, the transfer of a geneallele may also occur by recombination between two donor genomes in afused protoplast, wherein at least one donor protoplast carries thedesired gene allele in its genome. In each case the descendants, whichthen comprise the desired gene allele, can then be backcrossed againwith a line which comprises a preferred genetic background and can beselected for the desired gene allele. The result is fixing of thedesired gene allele in a selected genetic background.

The term “isolated nucleic acid molecule” or “isolate polynucleotide”should be understood to mean a nucleic acid molecule or polynucleotideremoved from its natural or original environment. The term alsoencompasses a synthetically produced nucleic acid molecule. An “isolatedpolypeptide” should be understood to mean a polypeptide which has beenremoved from its natural or original environment. The term alsoencompasses a synthetically produced polypeptide.

The term “pathogen infection” should be understood to mean the earliesttime at which a pathogen interacts with a plant host tissue. Examples infungi such as ascomycetes or oomycetes are the growth of hyphae or theformation of specific infection structures such as penetration hyphaeand the appressorium. In detail, an infection with Helminthosporiumturcicum may be investigated using various stain techniques (for exampletrypan blue) (Chung et al., BMC Plant Biology 10 (2010), 103; Walsh etal. (2008), Poster presentation P192, 50th Maize Genetics Conference inWashington D.C.).

“Donor Pepitilla”, “accession Pepitilla” or “Pepitilla” means, inaddition to the landrace Pepitilla itself, other maize genotypes intothe genome of which, in particular on chromosome 8 bin 5 or 6, anintrogression of the HTN1 resistance locus, preferably from Pepitilla,has been inserted. Examples of these are W22Htn (e.g. Bar-Zur et al.1998), H6314Htn (e.g. Bar-Zur et al. 1998), B73HtN (e.g. Shimoni et al.,Journal of Phytopathology 131:4 (1991), 315-321), B68HtN and A632HtN(e.g. Carson, Plant Disease 79 (1995), 717-720) and A619HtN (e.g.Stanković et al, Genetika 39:2 (2007), 227-240). Furthermore, Pepitillaincludes any source of resistance which confers the resistance phenotypewith the features typical of HTN1 after introgression into a vulnerablemaize line/maize plant. Examples of these HTN1-specific features aredelayed onset of sporulation, reduced development of lesions,development of smaller lesions, reduced sporulation zones and/or no oronly isolated chlorotic-necrotic lesions.

A “Locus” is a position on a chromosome where one or more genes arefound which cause an agronomic feature or influence one. In particular,“locus” as used here means the HTN1-resistance locus which confersresistance against the pathogen Helminthosporium turcicum or at leastagainst a race of Helminthosporium turcicum.

A “maize plant” should be understood to mean a plant from the speciesZea mays as well as its subspecies such as, for example, Zea mays ssp.mays, Zea mays ssp. mexicana or Zea mays ssp. parviglumis.

A “marker” is a nucleotide sequence which is used as a reference ororientation point. A marker for recognizing a recombination event shouldbe suitable for monitoring differences or polymorphisms in a plantpopulation. For markers, these differences are on a DNA level and, forexample, are polynucleotide sequence differences such as, for example,SSRs (simple sequence repeats), RFLPs (restriction fragment lengthpolymorphisms), FLPs (fragment length polymorphisms) or SNPs (singlenucleotide polymorphisms). The markers may be derived from genomic orexpressed nucleic acids such as spliced RNA, cDNA or ESTs and may bebased on nucleic acids which are used as probes or primer pairs and assuch are suitable for amplifying a sequence fragment using PCR-basedmethods. Markers which concern genetic polymorphisms between parts of apopulation can be detected using established methods from the prior art(An Introduction to Genetic Analysis. 7th Edition, Griffiths, Miller,Suzuki et al., 2000). These include, for example: DNA sequencing,PCR-based, sequence-specific amplification, assaying of RFLPs, assayingof polynucleotide polymorphisms using allele-specific hybridization(ASH), detection of SSRs, SNPs or AFLPs. Methods for detecting ESTs(expressed sequence tags) and RAPD (randomly amplified polymorphic DNA)are also known. Depending on the context, the term “marker” in thedescription may also mean a specific chromosome position in the genomeof a species where a specific marker (for example SNP) can be found. Amarker position of this type can be used in order to monitor thepresence of a coupled locus, for example a coupled locus whichcontributes to the expression of a specific phenotypical feature (e.g.HTN1 or linkage drag). As an example, the marker locus may also be usedto observe the segregation of alleles at a locus (QTL or individualgene) which are genetically or physically closely coupled with themarker position.

“Operatively linked” means linked in a common nucleic acid molecule in amanner such that the linked elements are positioned and orientated withrespect to each other such that transcription of the nucleic acidmolecule can take place. A DNA which is operatively linked with apromoter is under the transcriptional control of this promoter.

Examples of plant “organs” are leaves, plant stems, stems, roots,vegetative buds, meristems, embryos, anthers, ovulae or fruit. Plant“parts” means a fusion of several organs, for example a flower or a seedor a part of an organ, for example a cross segment from the stem.Examples of plant “tissues” are callus tissue, soft tissue, meristemtissue, leaf tissue, bud tissue, root tissue, plant tumour tissue orreproductive tissue. The term “cells” should be understood to meanisolated plant cells with a cell wall or aggregates thereof orprotoplasts, for example.

In the context of the invention, unless stated otherwise, a “plant” maybe any species of dicotyledon, monocotyledon or gymnosperm plant.Preferably, the plants are monocotyledon plants and are of interest inagriculture or horticulture or for the production of bioenergy(bioethanol, biogas, etc). Examples are Gossypium sp., Zea mays,Brachypodium distachyon, Triticum sp., Hordeum vulgare, Oryza sativa,Sorghum sp., Musa sp., Saccharum officinarum, Secale cereale, Avena sp.,turf grass and forage grass. A preferred plant in accordance with theinvention is a plant from the genus Zea, in particular the species Zeamays, or Sorghum.

In connection with the present invention, the term “regulatory sequence”means a nucleotide sequence which influences the specificity and/orstrength of expression, for example insofar as the regulatory sequenceconfers a specific tissue specificity. A regulatory sequence of thistype may be localized upstream of the transcription initiation point ofa minimum promoter, but also downstream thereof, for example in atranscribed but not translated leader sequence or within an intron.

The expression “resistance” or “resistant” as regards a pathogen shouldbe understood to mean the ability of a plant or plant cell to resist thedamaging effects of the pathogen and extends from a delay in thedevelopment of disease to complete suppression of the development of thedisease. In connection with the present invention, a plant/plant cell isresistant or a plant/plant cell has a resistance to the pathogenHelminthosporium turcicum (H. turcicum or HT), i.e. to the leaf diseaseNorthern Corn Leaf Blight (NCLB). The resistance is conferred by one ormore proteins which are coded by a gene or by genes(resistance-conferring genes) from the accession Pepitilla. Theresistance may be complete or partial and may be specific ornon-specific to the pathogen race. In the event of a pathogenrace-specific resistance, the virulent races of Helminthosporiumturcicum may, for example, include N, 1N, 2N, 23N or 123N; the avirulentraces may, for example, include 0, 1, 2, 3, 12, 23 or 123. A conferredresistance may be a newly inherited resistance or an increase in apartial resistance which is already extant.

A “transgenic plant” is a plant into the genome of which at least onepolynucleotide, preferably a heterologous polynucleotide, has beenintegrated. Preferably, the polynucleotide has been integrated in astable manner, which means that the integrated polynucleotide remainsstable in the plant, is expressed and can also be stably inherited todescendants. The stable introduction of a polynucleotide into the genomeof a plant also includes integration into the genome of a plant of theprevious parental generation, whereby the polynucleotide can be furtherinherited in a stable manner. The term “heterologous” means that theintroduced polynucleotide originates, for example, from a cell or anorganism with another genetic background of the same species or fromanother species, or is homologous with the prokaryotic or eukaryotichost cell, but then is localized in a different genetic environment andthus is different from any possible corresponding naturally occurringpolynucleotide. A heterologous polynucleotide can be present in additionto a corresponding endogenous gene.

Embodiments and variations of the present invention will now bedescribed with reference to the accompanying figures and sequences inwhich:

FIG. 1: Calculated QTL region of 23.11 cM on chromosome 8 using 8markers in 528 F2 individuals of the RP1×RP1 HTN1 cross. The black bars(HtN) show the confidence interval. Positions of the markers are in cM.

FIG. 2: Silage yield test on 5 locations in Germany and in twoduplications, with the recurrent parent RP3 and the A version of thedonor fragment from B37HTN1 (RP3HTNA) and the K version of the donorfragment from B37HTN1 (RP3HTNK). The bars show significant differencesusing the t-test, with p=0.05.

FIG. 3: Description of the marker regions M1 to M6 which define thechromosomal intervals (Int. 1 to Int. 5) which exhibit theresistance-conferring polynucleotide in the introgression lines andcarry linkage drag in the chromosome fragment originating from thedonor. Chromosomal segments of the donor (Pepitilla” are shown as dottedareas, those of the recurrent parent (without linkage drag) are shown asareas with diagonal stripes. Interval 1 (Int. 1) covers the resistancelocus HTN1, interval 2 (Int. 2) covers sequence regions which areresponsible in the donor for the linkage drag of the flowering time,intervals 4 and 5 (Int. 4 and Int. 5) cover sequence regions which areresponsible for linkage drag of the silage yield in the donor.

FIG. 4: BAC contig on its RP4HTN1 BAC bank with corresponding sequencescaffold and gene annotations. Candidate genes are shown in squaredboxes. The black arrows represent further annotated genes which are notcandidate genes for HTN resistance.

1. PHENOTYPING EXPERIMENTS

-   -   A) Carrying out field trials to determine the HT resistance        under natural and artificial inoculation/infection conditions        and the flowering time:        -   At a location, at least 20 individuals per maize genotype to            be investigated were planted out in a row. Inoculation was            carried out naturally or artificially. Natural            inoculation/infection was carried out using naturally            occurring spores of H. turcicum. Artificial            inoculation/infection was carried out using infected and            ground leaf material which was administered to the plants to            be tested. The latter type of inoculation allowed a            comparable H. turcicum infestation to be simulated in            different test years and at different locations            independently of the prevailing natural infestation            conditions there. A vulnerable parent and a parent with HTN1            introgression were cultivated from the donor B37HTN1 as            control genotypes, depending on the test cross population.            The classification score of the HT resistance feature was            noted at least three times during the vegetative period.            Only the classification score scheme shown in Table 3 was            used.        -   The donor B37HTN1 as the source of HT resistance was crossed            into various genetic backgrounds from elite lines with            various levels of vulnerability to H. turcicum and            near-isogenic lines were developed which were different from            the vulnerable original lines essentially only by the            introgression from B37HTN1. In phenotyping experiments,            after artificial inoculation as described above, lines were            selected which exhibited an improvement in the HT resistance            by at least 2 to 3 classification scores, preferably 3 to 4            classification scores by introducing the            resistance-conferring introgression from B37HTN1. The            present invention will be described below in more detail by            way of example using the two selected recurrent parents RP1            and RP3. The results for the phenotyping experiments            described are summarized in Table 5. The recurrent parent            RP1 without introgression exhibited average classification            scores of 7 to 9, which were improved by 3 to 4            classification scores by the introgression from B37HTN1. The            recurrent parent RP3 exhibited classification scores between            4 and 6 without introgression and an improvement of 2 to 3            classification scores by the introgression. The recurrent            parent RP4 exhibited a classification score of 6 without            introgression and an improvement of 2-3 classification            scores by the introgression.

TABLE 5 Phenotyping data for HT resistance from genotypes RP1, RP3, andRP4 with and without resistance conferred introgression from B37HTN1(classification scores were determined in accordance with the scheme inTable 3). Average classification scores (n = 20) Improvement in withoutHT resistance introgression from with introgression Genotype B37HTN1from B37HTN1 RP1 7 to 9 3 to 4 RP3 4 to 6 2 to 3 RP4 6 2 to 3

-   -   -   In addition to the HT resistance, for each genotype the time            of female and male flowering was determined as “days after            sowing”. The time for female flowering was determined by            silk emergence; of male flowering by the appearance of            panicles. The results are shown in more detail in Example            3.B).

    -   B) Carrying out field trials to determine grain and silage        yields:        -   In addition to the above data regarding HT resistance and            flowering time, yield data for RP3 containing different            lengths of resistance-conferring introgression fragments            from B37HTN1 or Pepitilla and for a comparative elite line            were determined. The lines RP3, RP3HTNA and RP3HTNK were            dusted with a tester (flint maize, interpool single cross)            of the complementary gene pool (flint maize) in order to            produce seed stock for test hybrids. These test hybrids were            each grown in duplicate in a field trial at five            representative locations for maize crops in Germany. The            test hybrids are well suited to these growing regions having            regard to ripening. The field trial was carried out in two            duplications in 4-row parcels 6 m in length and with a 0.75            m row separation. The density was 9 plants per m² in the            first and 11 plants per m² in the second duplication. At the            time of the silo maize harvest only the two central rows of            each parcel were harvested in order to minimize competition            effects. The weight per parcel and the water content were            determined for the harvested material in order to calculate            the silo maize yield (also known as the silage yield or the            total dry matter yield) and the dry matter content (total            dry matter content).

    -   C) Carrying out greenhouse trials in order to determine the HT        resistance:        -   20 individuals per genotype were grown in pots. The controls            were genotypes of a vulnerable parent and a near-isogenic            parent (NIL) with resistance-conferring introgression from            B37HTN1, depending on the cross. 14 days after sowing, an            artificial infection was carried out (see above). After a            further 2 to 3 weeks, the first symptoms of disease            developed. From the time of the appearance of the first            symptoms, every other day the classification scores of the            HT resistance feature as well as the number of plants with            symptoms were determined. From this, the AUDPC (area under            disease progress curve) was determined. The infestation            frequency (as the %/time×period) was used to classify the            plants under investigation; here, an AUDPC from 0-100 was            resistant, 101-450 was heterozygous, and >450 was            vulnerable.

2. MARKER DEVELOPMENT FOR THE HTN1-TARGET-REGION

-   -   In addition to the classification score tests, the target region        around the HTN1 resistance locus on chromosome 8 (bin 8.06) in        many genotypes was examined in more detail and mapped finely        using novel and/or optimized molecular markers. The molecular        markers used herein were developed on the basis of single        nucleotide polymorphisms (SNP) or already publically available        simple sequence repeat markers (SSR):    -   The DNA from the genotypes for use as markers was either        isolated using the NucleoSpin 96 Plant II method following the        manufacturer's instructions (MACHEREY-NAGEL GmbH & Co. KG,        Germany) or using the Klear Gene DNA Extraction 384 method (LGC        Genomics GmbH, Germany).    -   The primer sequences for the SSR markers were already known from        the public database from the National Center for Biotechnology        Information (NCBI) at http://www.ncbi.nlm.nih.gov/unists; the        primer sequences for the markers bnlg1782, umc1960, bnlg240,        umc1121, bnlg1067 and umc1287, which were used to examine the        target region, are summarized in Table 6, together with the        modifications made.

TABLE 6 Primer sequences for the SSR marker (NED: 2′-chloro-5′-fluoro-7′,8′-fused phenyl-1.4-dichloro-6-carboxyfluorescein; FAM:6-carboxyfluorescein; M13: core sequence for phage M13) Forward Reverseprimer primer sequence sequence Additional (5′-3′) (5′-3′) primer + [SEQModifi- [SEQ Modifi- modifi- Marker ID NO] cation ID NO] cation cationbnlg1782 113 NED 114 none umc1960 115 NED 116 none bnlg240 117 FAM 118none umc1121 119 FAM 120 none bnlg1067 121 FAM 122 none umc1287 123 none124 none M13 + FAM

-   -   The volume of the PCR reaction mixture of bnlg1782, umc1960,        bnlg240, umc1121 and bnlg1067 was 10 μl and consisted of a        single concentration of the 4× buffer B (Solis BioDyne,        Estonia), 0.5 pmol of the forward primer, 0.5 pmol of the        reverse primer, 10-30 ng of DNA, 0.25 units of HotFirepol        TAQ-Polymerase (Solis BioDyne, Estonia). The volume of the        reaction mixture of umc1287 was 10 μl and consisted of a single        concentration of the 4× bufferB (Solis BioDyne, Estonia), 0.5        pmol of the forward primer, 2.5 pmol of the reverse primer, 0.3        pmol of the additional primer M13, 10-30 ng of DNA, 0.25 units        of HotFirepol TAQ-Polymerase (Solis BioDyne, Estonia).    -   The PCR reaction was carried out with an initial denaturing        period of 900 seconds at 94° C., an amplification cycle of 25-40        cycles with 15 seconds at 94° C., 30 seconds at 50-55° C. and        120 seconds at 72° C., and a final step of 300 seconds at 72° C.        Next, the PCR reaction was incubated for 2 h at 65° C. The PCR        products were separated on an AB13730xl (Life Technologies™,        USA) following the manufacturer's instructions for the        separation of 50-400 bp fragments.    -   The SNP markers were developed and used either (a) from        publically available resources, (b) from a comparative amplicon        sequencing or (c) from a sequence comparison of BAC sequences        from RP4HTN1 (see Molecular Analysis segment) and B73 reference        genome AGPv02 (www.maizesequence.org).        -   (a) SNPs were transformed into KASP markers (LGC Genomics            GmbH, Germany) from the publically available SNP resource of            the Maize Community 50K-illumina-Chip (Ganal et al., 2011).            To this end, novel primers were developed which ensured the            amplification of the decisive alleles in the KASP marker            assay (see Table 4). The whole operation was carried out            using Kraken™ Software (LGC Genomics GmbH, Germany). For a            KASP marker assay, 5-20 ng DNA, 0.02 μl of an oligo assay            mixture (12 μM primer allele 1 (forward); 12 μM of primer            allele 2 (forward); 30 μM of reverse primer) and 1.5 μl of a            1×KASPar Reagent Kit for 1536 plates was used. A standard            PCR setup consisted of 94° C. for 15 min, 10 cycles at            94° C. for 20 seconds, 61-55° C. touchdown for 1 minute, 26            cycles at 94° C. for 20 seconds and 55° C. for 1 minute. The            evaluation of the alleles per genotype was carried out using            Kraken™ software (LGC Genomics GmbH, Germany).        -   (b) The comparative amplicon sequencing was carried out            using Sanger sequencing. The genotypes in the comparative            sequences each comprised the donor B37HTN1 as well as B37,            RP1, RP1HTN1, RP3, RP3HTN1 (versions A, B, K), RP4, RP4HTN1.            The DNA was isolated from ground grains using the CTAB            method (Maniatis et al., 1982). The primer sequences for the            amplicon sequencing are listed in Table 4. A standard PCR            protocol for amplification of the corresponding regions            consisted of denaturing at 94° C. for 5 minutes, 35 cycles            each at 94° C. for 1 minute, 60° C. for 1 minute and 72° C.            for 2 minutes and a subsequent step at 72° C. for 10            minutes. The PCR products were sequenced with the Sanger            method (Sanger & Coulson, 1975). The sequence evaluation was            carried out using DNAStar Lasergene software (DNASTAR Inc.,            USA). The detected polymorphisms were transformed into KASP            markers as described in (a).        -   (c) The BAC sequence contigs were projected against the B73            reference genome AGPv02 using Blast algorithms            (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in order to detect            single nucleotide polymorphisms (SNP). The polymorphisms            were detected using Lasergene software (DNASTAR Inc., USA)            and are shown in Table 4 along with the flanking sequences.            Primers were developed for the flanking sequences of an SNP            and the identified SNPs were transformed into KASP markers            as described in (a).

3. LOCALIZATION OF THE HTN1 RESISTANCE LOCUS ON CHROMOSOME 8 USING THESSR MARKER

-   -   A) Localization of the HTN1 resistance locus:        -   The HTN1 resistance locus from the B37HTN1 donors were            crossed into elite lines as described in Example 1.A) and            localized on chromosome 8 (bin 8.06) with the aid of the SSR            and SNP markers from Example 2 (see FIG. 1). NILs from the            crosses RP1×RP1HTN1 and RP3×RP3HTN1 were phenotyped at two            locations over several years with two duplications under            natural infection conditions using the classification score            scheme of Table 3. The NILs showed, on average, a resistance            response which was improved by 4 classification scores            compared with the original line. The development of local            lesions on the leaves was shifted by approximately 2 weeks            compared with the vulnerable genotype. QTL mapping was            carried out with 528 F2 individuals (RP1×RP1HTN1 cross)            using the 8 markers (Tables 4 and 6 are from the QTL mapping            markers of FIG. 1). The QTL region which covered the HTN1            resistance locus was localized on chromosome 8 between the            markers MA0002 and umc1287, in a 23.1 cM region.    -   B) Crossing the B37HTN1 donor fragment into an elite maize line        and identification and elimination of linkage drag for delayed        flowering time:        -   The donor B37HTN1 was crossed with KWS.elite, an elite maize            line from KWS SAAT AG (Germany) and then backcrossed over            five generations with KWS.elite. In each backcross            generation, molecular markers were used in order to select            plants which were heterozygous for the HTN target region.            Next, a selected plant from the fifth backcross generation            was self-fertilized and homozygous plants for the HTN target            region were identified with molecular markers.        -   These lines were tested in field trials at several            locations. In this regard, for the genotypes B37HTN1,            KWS.elite and KWS.elite.B37HTN1, the phenotypical data of HT            resistance and the flowering times were determined as            described in Example 1. The genotypes with HTN1            introgression exhibited the expected HT resistance with            classification scores of 1 to 3, while the original line            KWS.elite exhibited classification scores of 5-7.            Unexpectedly, in addition, compared with the KWS.elite, the            KWS.elite.B37HTN1 exhibited a flowering time both for the            female and for the male flowers which was shifted by at            least 2 days. These shifted flowering times constitute a            negative agronomic feature for maize based on linkage drag            which has not yet been described in this form following            introgression of HT resistance from B37HTN1. Marker analyses            found the localization of the linkage drag which is            responsible for the delayed flowering time to be in a region            between two marker regions on the introgression from            B37HTN1, between M1 and M2. In this regard, the genotypes            B37HTN1, KWS.elite and KWS.elite.B37HTN1 were, for example,            analysed with the KASP markers SYN14136, PZE-108076510,            SYN24931 and PZE-108077560 (see FIG. 3 and Table 4).            SYN14136 and PZE-108076510 were used for the specific            detection of the marker region M1, SYN24931 and            PZE-108077560 for the specific detection of the region M2.            According to this, the marker region M1 lies 5′ from the            locus of the linkage drag and the marker region M2 is 3′            thereto. The marker analysis showed that B37HTN1 and            KWS.elite.B37HTN1, both with a flowering delayed by two            days, exhibited common alleles for the regions M1 and M2 as            well as the interval between these regions, while KWS.elite            has a normal flowering time and has other alleles for the            regions M1 and M2 and the interval between them.        -   The donor B37HTN1 was crossed with RP3 and then backcrossed            over three generations with RP3. Molecular markers were used            in each backcross generation. Initially, plants which were            heterozygous for the HTN1 target region were selected and            then these plants were investigated with markers which were            distributed uniformly over the genome in order to select            against the donor genome. Next, a selected plant from the            third backcross generation was self-fertilized and            homozygous plants for the HTN1 target region were identified            with molecular markers.        -   Furthermore, the donor B37HTN1 was also crossed with the            recurrent parent RP3 and RP4 and a RP3HTNA and RP4HTNA line            produced over several backcrossing steps. The phenotyping on            HT resistance showed an improvement in the classification            scores of 5 to 7 for the original line RP3 to 1 to 3 for            RP3HTNA and an improvement in the classification scores from            6 for the original line RP4 to 2 to 3 for RP4HTNA. The            phenotyping for flowering time exhibited comparable            flowering times for RP3 and RP3HTNA and RP4 and RP4HTNA.            Using the KASP markers SYN14136, PZE-108076510, SYN24931 and            PZE-108077560 showed that RP3 and RP3HTNA carry common            alleles for the regions M1 and M2. These did not correspond            to the donor B37HTN1. As a result, then, the flowering            time-delaying chromosomal segment of the introgression from            B37HTN1 lies on a chromosomal interval between the marker            regions M1 and M2. With the line RP3HTNA, then, this linkage            drag was successfully removed. The KASP markers used,            SYN14136, PZE-108076510, SYN24931 and PZE-108077560, proved            to be effective tools for “assisted selection”.        -   Phenotyping of RP3 and RP3HTNA also comprised recording the            grain and silage yield. While the grain yield in the            genotypes was not significantly different, the silage yield            feature in RP3HTNA exhibited an unequivocal, statistically            significant reduction of at least 14 decitonnes per hectare            (dt/ha) over RP3, or a reduction of more than 5%.        -   With the aid of the designed KASP markers SYN14136,            PZE-108076510, SYN24931 and PZE-108077560, a RP1HTN1 line            could be selected from the cross of B37HTN1 and the            recurrent parent RP1 which did not exhibit any more            flowering time-delaying linkage drag, but rather a silage            yield reduction, as was observed for RP3HTNA. For the            purposes of more accurate molecular characterization,            RP1HTN1 was developed further and a F2 population was set up            with 724 individuals from the cross RP1×RP1HTN1. Next, the            F3 generation was self-fertilized and selected F4 plants            were genotyped and phenotyped. Genotyping was carried out            using markers from Table 6 in the detected QTL region of            23.1 cM. Phenotyping was carried out at several locations in            two duplications (see Example 1). Recombinant plants for the            QTL region were selected and correlated with the phenotype            data. The selection comprised plants which covered different            regions of the target region as well as heterozygous plants,            with the aim of obtaining new recombinant plants. Each year,            two backcrosses with RP1 were carried out and individual            plants were selected, and thus new recombinants were            produced. New recombinants were phenotyped in field and            greenhouse tests (see under 1.) and genotyped for the            development of novel molecular markers in accordance with 2.        -   The use of these novel markers on the RP3HTNA genotype            allowed a marker region M3 to be identified which limited            the introgression in the 5′ region and can be described with            the flanking markers PZE-108093423 and PZE-108093748. In            this regard, PZE-108093423 should exhibit the alleles of the            recurrent parent RP3 and PZE-108093748 should exhibit the            alleles of the donor B37HTN1. In the 3′ region, the            introgression of RP3HTNA by the markers PZE-108107671 and            SYN4196 in a further marker region M6 is described (see FIG.            3). In this regard, PZE-108107671 carries the alleles of the            donor B37HTN1 and SYN4196 carries the alleles of the            recurrent parent RP3. The introgression from RP3HTNA            (hereinafter termed version        -   A) corresponds, between the marker regions M3 and M6, to the            donor B37HTN1, but outside this region it corresponds to the            recurrent parent or another line which does not carry the            alleles in the region of the donor B37HTN1 between M1 and            M2. This version A was introduced into various other genetic            backgrounds and fresh yield tests, resistance phenotyping            and flowering time determinations were undertaken. The            results were comparable with those described for RP3HTNA.            Thus, the flowering time was not shifted compared with the            corresponding line without introgression and the line            exhibited an improved resistance to Helminthosporium            turcicum compared with the original line, or at least the            reduction of the silage yield.    -   C) Identification and elimination of linkage drag for reduced        silage yield:        -   The donor used was the RP3HTNA line. This was crossed with            RP3 and self-fertilized over six further generations. In            each self-fertilization generation, molecular markers were            used in the target region in order to reduce the donor            fragment. Since all regions of the genome outside the target            region had already been selected in the RP3HTNA line on the            RP3 genome, only the region around the HTN target region was            investigated with markers. In this regard, homozygous plants            were identified for a reduced HTN target region. At the same            time, intensive marker development was carried out in the            target region. In addition to many others, a RP3HTNK line            was identified which described the B37HTN1 donor fragment            from a marker region M4 flanked by the markers MA0004 and            MA0005, wherein MA0004 describes the alleles of the            recurrent parent RP3 and MA0005 describes the alleles of the            donor B37HTN1 in RP3HTNK, up to a marker region M5, flanked            by the markers MA0006 and PZE-108097482, wherein MA0006            describes the alleles of the donor B37HTN1 and PZE-108097482            describes the alleles of the recurrent parent RP3. In            RP3HTNK, the introgression from RP3HTNK (hereinafter termed            version K) causes an improved HTN1 resistance of 3 to 4            classification scores compared with RP3, the same flowering            time as its original line RP3 (no delay in flowering) and no            further significant reduction in the silage yield (see FIG.            2). In addition, with the aid of the described markers,            linkage drag-free lines could be produced from the original            line RP1 by crossing, which lines exhibited version K of the            introgression.    -   D) Resistance-conferring haplotype from B37HTN1 or from        Pepitilla        -   Version K possesses a haplotype from B37HTN1 or from            Pepitilla which carries the donor alleles described in Table            4 at the physical positions with respect to B73 AGPv02 in            bp. As an example, the haplotype at marker MA0008 will be            described: using the marker MA0008 and specifying the            alleles for B37HTN1, RP3, RP3HTNA, RP3HTNK, then the allele            “T” is for B37HTN1, RP3HTNA, RP3HTNK and the allele “C” is            for RP3. For this locus, this marker also distinguishes the            assumed HTN1 resistance source PH99N (WO 2011/163590), which            also contains an allele “C” at this position, from the            resistance source used here.

4. MOLECULAR ANALYSIS OF THE FINE-MAPPED REGION

Furthermore, the chromosome fragment which had been inserted andtruncated by introgression was investigated on a molecular level. Theresistance locus Htn1 from the accession Pepitilla was thus reduced to adistinct target region, a chromosome interval of 700 kb, and sequencedin the genotype RP4HTN1. As will be described in more detail below, BACclones from RP4HTN1 were isolated, sequenced and assembled into asequence scaffold. The sequence scaffold was annotated and the annotatedgenes in this interval were set against EST/cDNA sequence information.Differential expression studies were then carried out from amultiplicity of annotated genes to identify the candidate genes (seeTable 1).

-   -   A) BAC bank and pool construction, BAC bank screening, BAC        sequencing        -   A BAC bank was produced from the genotype RP4HTN1. This was            followed by constructing the BAC bank and the 3D matrix pool            from leaf material as well as by screening the 3D matrix            pool. The primers for screening the 3D matrix pool were            based on the B73 AGPv01 sequence from 149957158 bp to            152977351 bp on chromosome 8 (www.maizesequence.org) and the            primer program 3 (http://simgene.com/primer3; Rozen &            Skaletsky, 2000). The parameters for the primer selection            were a mean GC content of 50%, primer length of 20-25 bp,            melting temperature between 70-90° C. and amplicon length            between 70-80 bp. Using the primer pairs in Table 7, the 3D            pools were screened using RT-PCR. The values of the two            parameters, namely melting temperature and CP value, are            given for the BAC clone. 26 BAC clones could be identified            for the selected region. All BAC clones were isolated from            the BAC bank and used as E. coli culture for DNA isolation            and sequencing. Sequencing was carried out with a standard            GS-FLX titanium kit (454 Life Sciences, USA). The sequence            information obtained for the BAC clones 144N24, 119F13,            219G11, 86N21, 16B06, 84L18, 128D02, 25M23, 96H10, 19J24,            136A01, 75H06, 135F07 is summarized in Table 8.

TABLE 7 Primer pairs for detection of BAC clones from the RP4HTN BACbank Melting temp, CP value ° C. (50% (cycle of amplicon when the issingle exponential BAC Sequence, stranded) phase of clone Primer pair 1primer pair in genotype the PCR Amplicon ID Primer pair 2 1 (5′-3′)RP4HTN1 begins) size (bp) 58A14 579ZMPM0_2F; 125; 77.4 28.5 74579ZMPM0_2R 126 579ZMPM0_4F; 127; 80.96 26.52 77 579ZMPM0_4R 128 144N24579ZMPM0_5F; 129; 79.09 27.09 76 579ZMPM0_5R 130 579ZMPM0_17F; 131;83.06 25.53 78 579ZMPM0_17R 132 219G11 579ZMPM0_16F; 133; 84.7 25.96 78579ZMPM0_16R 134 579ZMPM0_25F; 135; 78.95 26.09 80 579ZMPM0_25R 136119F13 579ZMPM0_22F; 137; 80.89 25.98 73 579ZMPM0_22R 138 579ZMPM0_34F;139; 80.1 24.43 76 579ZMPM0_34R 140 86N21 579ZMPM0_35F; 141; 80.9 25.2770 579ZMPM0_35R 142 579ZMPM0_38F; 143; 83.86 26.01 71 579ZMPM0_38R 14416B6 579ZMPM0_37F; 145; 79.22 25.71 80 579ZMPM0_37R 146 579ZMPM0_41F;147; 75.93 26.6 74 579ZMPM0_41R 148 84L18 579ZMPM0_41F; 149; 75.93 26.674 579ZMPM0_41R 150 579ZMPM0_46F; 151; 80.54 25.68 78 579ZMPM0_46R 152128D2 579ZMPM0_180F; 153, 84.41 25.99 77 579ZMPM0_180R2 154579ZMPM0_48F; 155; 83.96 25.33 77 579ZMPM0_48R 156 25M23 579ZMPM0_48F;157; 83.96 25.33 77 579ZMPM0_48R 158 579ZMPM0_56F; 159; 77 29.12 79579ZMPM0_56R 160 19J24 579ZMPM0_51F; 161; 87.76 27.75 77 579ZMPM0_51R162 579ZMPM0_199F; 163; 82.49 26.56 79 579ZMPM0_199R 164 96H10579ZMPM0_63F; 165; 85.78 26.08 63 579ZMPM0_63R 166 579ZMPM0_208F; 167;79.87 26.84 79 579ZMPM0_208R 168 136A1 579ZMPM0_206F; 169; 89.81 32.0970 579ZMPM0_206R 170 579ZMPM0_86F; 171; 81.81 30.07 71 579ZMPM0_86R 172135F7 579ZMPM0_79F; 173; 75.82 25.43 72 579ZMPM0_79R 174 579ZMPM0_278F;175; 78.13 22.69 78 579ZMPM0_278R 176 75H6 579ZMPM0_209F; 177; 75.4124.93 77 579ZMPM0_209R 178 579ZMPM0_86F; 179; 81.81 30.07 71579ZMPM0_86R 180 117O2 579ZMPM0_87F; 181; 81.89 27.7 76 579ZMPM0_87R 182579ZMPM0_91F; 183; 80.13 26.93 75 579ZMPM0_91R 184 173H23 579ZMPM0_216F;185; 82.3 25.76 80 579ZMPM0_216R 186 579ZMPM0_95F; 187; 79.5 24.97 73579ZMPM0_95R 188 118N19 579ZMPM0_99F; 189; 76.84 24.69 80 579ZMPM0_99R190 579ZMPM0_244F; 191; 80.07 25.38 80 579ZMPM0_244R 192 42L23579ZMPM0_241F; 193; 81.16 25.79 79 579ZMPM0_241 R 194 579ZMPM0_109F;195; 77.89 25.28 74 579ZMPM0_109R 196 112N13 579ZMPM0_109F; 197; 77.8925.28 74 579ZMPM0_109R 198 579ZMPM0_247F; 199; 80.76 24.82 71579ZMPM0_247R 200 97K23 579ZMPM0_112F; 201; 79.22 25.2 77 579ZMPM0_112R202 579ZMPM0_125F; 203; 83.44 28.17 74 579ZMPM0_125R 204 18J17579ZMPM0_253F; 205; 77.5 32.34 71 579ZMPM0_253R 206 579ZMPM0_125F; 207;83.44 28.17 74 579ZMPM0_125R 208 5M22 579ZMPM0_128F; 209; 77.99 24.05 77579ZMPM0_128R 210 579ZMPM0_136F; 211; 78.65 26.46 78 579ZMPM0_136R 212146I6 579ZMPM0_131F; 213; 76.58 26.54 78 579ZMPM0_131R 214579ZMPM0_137F; 215; 83.7 25.42 73 579ZMPM0_137R 216 147O15579ZMPM0_138F; 217; 79.38 24.8 79 579ZMPM0_138R 218 579ZMPM0_147F; 219;79.63 26.77 80 579ZMPM0_147R 220 88K17 579ZMPM0_145F; 221; 81.51 27.6176 579ZMPM0_145R 222 579ZMPM0_262F; 223; 75.7 25.82 80 579ZMPM0_262R 224180G22 579ZMPM0_161F; 225; 80.21 25.16 73 579ZMPM0_161R 226579ZMPM0_265F; 227; 79.3 24.7 79 579ZMPM0_265R 228

TABLE 8 Sequence content of the 13 analysed BAC clones # Reads Sequencequantity without Sequence quantity in bp without BAC # Reads E. coli inbp E. coli 144N24 10967 10849 3646226 3591222 119F13 17987 17847 60339105957456 219G11 32904 32484 10553629 10381924 86N21 39606 39106 1299159612750077 16B06 36198 35849 12523123 12357036 84L18 50537 34162 1599164510776458 128D02 15998 15847 5138442 5064677 25M23 22551 22416 78644937786402 96H10 7723 7614 2569604 2525488 19J24 21953 21775 73273647234315 136A01 31998 31724 10298869 10158900 75H06 24345 24121 80217277920125 135F07 29702 29484 9721708 9604010

-   -   B) BAC sequence assembly, annotation and candidate gene        selection:        -   Production of a sequence scaffold: the BAC clones 144N24,            119F13, 219G11, 86N21, 16B06, 84L18, 128D02, 25M23, 19J24,            96H10, 136A01, 75H06, 137F07 were sequenced using the 454            technique (Margulies et al., 2005).        -   Automatic assembly of the raw sequences of the BAC clones            was carried out with the “Newbler” software (454 runAssembly            software, software release 2.3). The pro BAC sequence            contigs produced in this manner were arranged correctly by            manual analysis, in which the following techniques were            applied:        -   1. Sequences of overlapping BACs could be roughly divided            into overlapping and non-overlapping zones.        -   2. Sequence contigs from various overlapping BACs were            compared in the overlapping zones. Approximately 20% of the            sequence contigs could be arranged in this manner and gaps            between them could be closed (for example when a contig of            one BAC covered or connected to two contigs of the other            BACs).        -   3. All sequence contigs were manually annotated. In this            regard, initially only repetitive elements (transposons and            retrotransposons, abbreviated to “TEs”) were annotated.            Since sequence gaps occur primarily in TEs, the TE            annotation can help to correctly arrange sequence contigs.            This means that when one end of a TE is on one sequence            contig and the other end is on another, the two contigs can            be ordered appropriately. In such cases, a sequence of 100            Ns is respectively inserted in order to fill the gaps            between the sequence contigs. In addition, the information            from TEs which are nested (i.e. TEs which have been inserted            into other TEs) was used in order to arrange sequence            contigs.        -   4. In some zones, neither information from overlapping BACs            nor TE annotations could be used (this was the case, for            example, in zones which were only covered by one BAC). Here,            the sequence contigs were arbitrarily arranged and the gaps            between them filled with sequences of 200 Ns.        -   5. Many of the TEs in the maize genome are “long terminal            repeat” (LTR) retrotransposons which are flanked by long            (1-2 kb) LTR sequences. These LTRs may be up to 100%            identical. In some cases, then, raw sequences of the two            LTRs were assembled into a consensus sequence (i.e. a copy            of the LTR is not present in the assembly). In these cases,            the sequence gaps were filled with the number of Ns which            would correspond to the length of the second LTR.        -   6. Genes were manually annotated. To this end, the coding            sequences (CDS) for the published B73 maize genome was used            as the reference (http://www.maizegdb.org/gene_model.php).            The CDS were aligned with the RP4HTN sequence using DotPlot            (http://www.dotplot.org/) and so the positions of exons and            introns were determined. Candidate genes were on the one            hand determined by describing their function (if publically            known). On the other hand, the CDS of the resistant RP4HTN            was compared with the vulnerable B73 AGPv02. If differences            occurred, the respective gene was placed in the list of            candidates. The prepared sequence had a length of 1′328′253            bp. The list of candidate genes is given in Table 1.

5. MOLECULAR ANALYSIS OF THE CANDIDATE GENES

Expression analysis: the expression of the various candidate genes wastested on 21 day old (following sowing), uninfected plants (infectionday=0 dpi) and also at 36 days old with plants which had been infectedand also which had not been infected with H. turcicum (15 days afterinfection=15 dpi inf/ni).

RNA from the second leaf was extracted from the tested maize plants,reverse transcribed into cDNA and the expression was measured usingqPCR. In each case the second leaf was harvested, frozen and the RNA wasextracted, quantified and tested for quality and purity using the SVTotal RNA Isolation System Kit (Z3100; Promega, Dübendorf, Switzerland),exactly as described (Brunner et al., 2011; Risk et al., 2012). 1 μg ofRNA was reverse transcribed using the iScript RT Supermix (170-8841;Bio-Rad, Cressier, Switzerland) in a reaction volume of 20 μl, followingthe manufacturer's instructions. In order to exclude the possibility ofcontamination by genomic DNA (RT minus), at the same time, a reactionwithout adding the reverse transcriptase was incubated for each sample.

Quantitative Real Time PCR (RT-qPCR) was carried out in technicaltriplicate or duplicate in a reaction volume of 10 μl and with theaddition of 4 μl of 1:10 diluted (10 mM Tris HCL pH8, 0.1 mM EDTA) cDNA,5 ul of SsoFast EvaGreen® Supermix (172-5201; Bio-Rad, Switzerland) anda primer concentration of 400 nM on the C1000 Touch Cycler (Bio-Rad,Switzerland). For amplification, the following program was used: 95° C.for 30 seconds, followed by 40 cycles at 95° C. for 3 seconds, then60-63° C. (see Table 2) for 5 seconds. To analyse the melting curve(exclusion of primer dimers), the PCR product was heated in 0.5° C.steps from 65° C. to 95° C. Amplification curves and melting curves werechecked in the CFX Manager V 3.0 (Bio-Rad, Switzerland) program and theCq values (quantification cycle) were exported into the qbasePLUS V 2.3(Biogazelle, Zwijnaarde, Belgium) program to determine the relativeexpressions.

The primers for the candidate genes were determined with the aid ofprimer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), inorder, as far as possible, to exclude non-specific amplification ontranscripts which were already known. In order to evaluate suitableamplicons, the PCR products were separated using agarose gelelectrophoresis and their sizes were examined using isolated bands.Furthermore, amplicons from RP1 HtN and also NILHtN as set out in Table1 were sequenced. In order to normalize the expression data, 1-3reference genes (LUG, MEP, FPGS) were used (Manoli et al., 2012). All ofthe candidate genes were expressed in the vulnerable genotype RP1 and inthe resistant genotype RP1 HTN. A differential expression between RP1and RP1 HTN could be demonstrated for RLK1. RLK1 in the vulnerableplants is expressed up to 4 times more strongly than in the resistantplants.

TABLE 9 Primer pairs for candidate genes, with their ampliconlength in bp and the appropriate annealing temperature. SEQPrimer sequences (F = Length Length Gene Primer ID Forward sequence; R =(in bp) (in bp) in Annealing name name NO: Reverse sequence) in RP1RP1HTN temperature ZNF1 GH034 229 F: TGGTTGGTGTCGAAGCTGAG 130 13060° C.  GH033 230 R: ATTTATCCCGGCCTTTGCAT HYD GH039 231F: GATCTACAGGGAAGCCCACTGA 74 74 60° C.  GH040 232R: TTTTTCCTTGAGGCAGTTATATGCT RLK4 GH220 233 F: TTGTGCAGCGGAGGGAA 91 8563° C.  GH221 234 R: CCAGGGCACCAGCAAGAAT EXT1 GH168 235F: CGACTACAAGACGCGTACC 103 103 60° C.  GH170 236 R: GGTGTCGATGGTGAGGTTCRLK1 GH138 237 F: TATTGTTGGTGCTGTTGCCG 121 121 60° C.  GH139 238R: GGACTCAATCCTTGTCCCTG RET1 GH055 239 F: CGCTCGTTTGCCAGATAGCC 165 16560° C.  GH056 240 R: CACGGTGTGTGCCAGTTTGT

TILLING population screening and detection of mutants: For the candidategenes (Table 1), screening of a TILLING population of 10000 plants whichcarries the introgression from Pepitilla on chromosome 8 in the regionfrom Ser. No. 15/688,552-153139596 bp compared to the B73 referenceAGPv02 (www.maizesequence.org) (RP3HTN1) and which exhibits a resistanceto Helminthosporium turcicum was carried out. The mutations could beeither silent nucleotide exchanges, amino acid exchanges or stop codonsand acted to detect the function of the candidate genes.

Transformation: Candidate genes could, for example, be introduced intothe vulnerable genotype A188 by means of Agrobacteriumtumefanciens-conferred transformation. This genotype is characterized byAUDPC values of 702 in the GWH-Test (n=18 plants), so that atransformation with the resistance gene produces a clear resistanceresponse.

6. DETERMINATION OF RACE SPECIFICITY: PROOF THAT HTN1 ALSO CONFERSRACE-NON-SPECIFIC RESISTANCE

Screening of the genotypes with the HtN gene was carried out at manylocations in all of the infestation regions of Europe. Until now, thisresistance has not been broken, so that we started with the assumptionthat until now they were not race-specific until a race N was found.Race 1 predominates in Europe, but in some individual regions, races 2or 3 or a combination thereof could be detected (Hanekamp et al., 2013).

7. PHENOTYPE TEST ON OTHER RECOMBINATION PLANTS

New recombination plants were tested for the QTL region and correlatedwith the phenotype data. The selection comprised plants which covereddifferent regions of the target region. Recombinant plants could beidentified which exhibited an introgression of the donor B37HTN1 betweenthe markers MA0005 and MA0021—marker region M7 and the markers MA0013and MA0022—marker region M8, in the genetic background of RP1. FIG. 4shows that this region only comprises the three genes RLK4, EXT1 andRLK1. These recombination plants, which comprise the region M7-M8,exhibit the resistance phenotype both in the field with artificialinoculation and also in the greenhouse test.

8. IDENTIFICATION OF THE RESISTANCE-CONFERRING CANDIDATE GENE

In order to identify the resistance-conferring gene, screening of theTILLING population of 10000 plants which exhibited the introgressionfrom Pepitilla on chromosome 8 in the region from Ser. No.15/688,552-153139596 bp compared with the B73 reference AGPv02(http://www.genome.arizona.edu) (RP3HTN1) and a resistance toHelminthosporium turcicum was carried out.

Amplicons were developed for genes RLK4 and RLK1 (Table 10) and afteramplification of the individual plant DNA of the TILLING population,these were sequenced by means of Sanger sequencing.

TABLE 10 Primer sequences for amplicons SEQ Primer sequences Length ofAnnealing Gene Primer ID (F = Forward sequence; amplicons temperaturename name NO: R = Reverse sequence) (in bp) (° C.) RLK4 MA04916-6f 247F: TGTTTCAGGAATCACGCAACTGGA 399 60 MA04916-6r 248R: GCACCACGCCATGACCAACATC RLK1 TG10013-10.f 249F: CTTCCTACAGAAGAACGAGAGT 804 60 TG10013-11.r 250R: TTCCTCACGAGCTCTGTGGTC

The amplicon sequences were evaluated using DNASTAR Lasergene softwareand base mutations were identified. Table 11 lists a selection of themutations found.

TABLE 11 Identified mutations for the genes RLK4 and RLK1 Position ofPosition of mutation the mutation in protein Amino in cDNA of sequenceof acid Gene Mutation RP3HTN1 Base RP3HTN1 exchange name name (bp)exchange (bp) effect RLK4 RLK4d 977 in SEQ G > A 326 in SEQ G > D ID NO:3 ID NO: 4 RLK4f 1169 in SEQ C > T 390 in SEQ T > I ID NO: 3 ID NO: 4RLK1 RLK1b 1365 in SEQ G > A 455 in SEQ M > I ID NO: 1 ID NO: 2 RLK1d1490 in SEQ G > A 497 in SEQ G > E ID NO: 1 ID NO: 2

The identified mutants were self-fertilized in the greenhouse and seedstock was harvested from the homozygous plants with the wild type alleleand mutation allele per mutation event for a phenotype test.

15 homozygous individual plants with a wild type allele and mutationallele for the mutants RLK1b, RLK1d, RLK4d and RLK4f and the controlsRP1 and RP1HTN1 were inoculated with H. turcicum as described above, ina greenhouse. In the period from 11 to 25 days following inoculation,the infestation was determined every day. The AUDPC values for all ofthe test plants are summarized in Table 12. Changing the amino acid inthe resistant parent of the RP3HTN1 TILLING population was expected tomake the homozygous mutants vulnerable.

TABLE 12 AUDPC values for homozygous plants with wild type allele andmutation allele of the genes RLK1 and RLK4. In the phenotype column,0-100 means resistant, 101-450 means heterozygous, and >450 meansvulnerable. Mutant name Alleles AUDPC Phenotype RLK4d Hom. Mutant 33.3resistant Hom. Wild type 0.0 resistant RLK4f Hom. Mutant 46.7 resistantHom. Wild type 96.7 resistant RLK1b Hom. Mutant 346.7 heterozygous Hom.Wild type 46.4 resistant RLK1d Hom. Mutant 406.7 heterozygous Hom. Wildtype 83.3 resistant RP1 1030.0 vulnerable RP1HTN1 0.0 resistant

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1-15. (canceled)
 16. A method for identifying a Helminthosporiumturcicum-resistant maize plant, the genome of which maize plant has achromosome fragment from a donor Pepitilla integrated therein,comprising: detecting at least two alleles in the genome of the plant;(i) wherein at least one allele is localized in a genomic segment whichis flanked by a marker in a first marker region, a second marker region,a third marker region, or a fourth marker region and by a polynucleotidewhich confers resistance against Helminthosporium turcicum in the maizeplant; and (ii) wherein at least one allele is localized in a genomicsegment which is flanked by the polynucleotide which confers resistanceagainst Helminthosporium turcicum and by a marker in a sixth markerregion or a fifth marker region.
 17. The method as claimed in claim 16,wherein: the first marker region is flanked by the markers SYN14136 andPZE-108076510, the second marker region is flanked by the markersSYN24931 and PZE-108077560, the third marker region is flanked by themarkers PZE-108093423 and PZE-108093748, the fourth marker region isflanked by the markers MA0004 and MA0005, the fifth marker region isflanked by the markers MA0006 and PZE-108097482, and the sixth markerregion is flanked by the markers PZE-108107671 and SYN4196.
 18. Themethod as claimed in claim 16, wherein the identification of aHelminthosporium turcicum-resistant maize plant comprises the use of anoligonucleotide which comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 17-250.
 19. The method as claimed inclaim 16, wherein the identification of a Helminthosporiumturcicum-resistant maize plant comprises the use of an oligonucleotidewhich comprises a nucleotide sequence selected from the group consistingof SEQ ID NOs: 41-49, 53-100, and 229-250, wherein the oligonucleotidecorresponds to at least one of the first marker region which is flankedby the markers SYN14136 and PZE-108076510, the second marker regionwhich is flanked by the markers SYN24931 and PZE-108077560, the thirdmarker region which is flanked by the markers PZE-108093423 andPZE-108093748, the fourth marker region which is flanked by the markersMA0004 and MA0005, the fifth marker region, which is flanked by themarkers MA0006 and PZE-108097482, and the sixth marker region which isflanked by the markers PZE-108107671 and SYN4196.
 20. A maize plantidentified by the method of claim 16, wherein the chromosome fragmentcomprising the interval of the donor comprises the polynucleotide whichconfers resistance to Helminthosporium turcicum.
 21. A cell, a tissue,or a part of the maize plant as claimed in claim 20, the cell, tissue,or part comprising the chromosome fragment comprising the interval ofthe donor which comprises the polynucleotide which confers resistance toHelminthosporium turcicum in the maize plant.
 22. A grain or a seed fromthe maize plant as claimed in claim 20, the grain or seed comprising thechromosome fragment comprising the interval of the donor which comprisesthe polynucleotide which confers resistance to Helminthosporium turcicumin the maize plant.
 23. The method as claimed in claim 16, wherein thechromosome fragment comprising the interval of the donor furthercomprises the interval of the donor which exhibits at least the donorallele of the marker MA0008.
 24. The method as claimed in claim 16,wherein the polynucleotide which confers resistance to Helminthosporiumturcicum comprises: a) comprises a nucleotide sequence in accordancewith SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, b) comprising a nucleotidesequence with an identity of at least 80% with one of the nucleotidesequences in accordance with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15,preferably over the entire length of the sequence, c) which codes for apolypeptide with an amino acid sequence in accordance with SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or 16, or d) which codes for a polypeptide with anamino acid sequence which has at least 80% identity with one of theamino acid sequences in accordance with (d).
 25. The method as claimedin claim 16, wherein the chromosome fragment: a) does not comprise aninterval of the donor between a marker in the first marker region whichis flanked by the markers SYN14136 and PZE-108076510 and a marker in thesecond marker region which is flanked by the markers SYN24931 andPZE-108077560, and/or b) does not comprise an interval of the donorbetween a marker in the third marker region which is flanked by themarkers PZE-108093423 and PZE-108093748 and a marker in the fourthmarker region which is flanked by the markers MA0004 and MA0005, and/orc) does not comprise an interval of the donor between a marker in thefifth marker region which is flanked by the markers MA0006 andPZE-108097482 and a marker in the sixth marker region which is flankedby the markers PZE-108107671 and SYN4196.
 26. The method as claimed inclaim 16, wherein the chromosome fragment: a) does not comprise aninterval of the donor defined by the marker SYN14136 and the markerSYN24931; b) does not comprise an interval of the donor defined by themarker PZE-108093748 and the marker MA0005; and c) does not comprise aninterval of the donor defined by the marker MA0006 and the markerPZE-108107671,
 27. The method as claimed in claim 16, wherein thechromosome fragment does not comprise an interval of the donor definedby and including the marker SYN14136 and the marker SYN24931, andwherein the markers SYN14136 and SYN24931 are part of the interval. 28.The method as claimed in claim 16, wherein the chromosome fragment doesnot comprise an interval of the donor defined by and including themarker PZE-108093748 and the marker MA0005, and wherein the markersPZE-108093748 and MA0005 are part of the interval.
 29. The method asclaimed in claim 16, wherein the chromosome fragment does not comprisean interval of the donor defined by and including the marker MA0006 andthe marker PZE-108107671, and wherein the markers MA0006 andPZE-108107671 are part of the interval.